Molecular markers linked to ppo inhibitor tolerance in soybeans

ABSTRACT

This invention relates generally to the detection of genetic differences among soybeans. More particularly, the invention relates to soybean quantitative trait loci (QTL) for tolerance to protoporphyrinogen oxidase inhibitors, to soybean plants possessing these QTLs, which map to a novel chromosomal region, and to genetic markers that are indicative of phenotypes associated with protoporphyrinogen oxidase inhibitor tolerance. Methods and compositions for use of these markers in genotyping of soybean and selection are also disclosed.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of application Ser. No.12/506,498, filed Jul. 21, 2009, which claims priority under 35 U.S.C.§119(e) to provisional application Ser. No. 60/083,038 filed Jul. 23,2008, herein incorporated by reference in their entirety.

FIELD OF THE INVENTION

This invention relates generally to the detection of genetic differencesamong soybeans.

BACKGROUND OF THE INVENTION

Soybeans (Glycine max L. Merr.) are a major cash crop and investmentcommodity in North America and elsewhere. Soybean oil is one of the mostwidely used edible oils, and soybeans are used worldwide both in animalfeed and in human food production. Additionally, soybean utilization isexpanding to industrial, manufacturing, and pharmaceutical applications.Weed management in soybean fields is important to maximizing yields. Arecent development in soybean technology has been the development ofherbicide-tolerant soybean varieties. Glyphosate tolerant soybeans werecommercially introduced in 1996 and accounted for more than 85% percentof U.S. soybean acreage in 2007.

Some weeds are starting to show increased tolerance to glyphosate. Thisincreased tolerance decreases the effectiveness of glyphosateapplication and results in lower yields for farmers. As a result thereis a need in the art for soybean varieties that are tolerant to otherherbicide chemistry.

SUMMARY OF THE INVENTION

This invention relates generally to the detection of genetic differencesamong soybeans. More particularly, the invention relates to soybeanquantitative trait loci (QTL) for tolerance to protoporphyrinogenoxidase (PPOase) inhibitors, to soybean plants possessing these QTLs,which map to a novel chromosomal region, and to genetic markers that areindicative of phenotypes associated with protoporphyrinogen oxidaseinhibitor tolerance. Methods and compositions for use of these markersin genotyping of soybean and selection are also disclosed.

A novel method is provided for determining the presence or absence insoybean germplasm of a QTL associated with tolerance toprotoporphyrinogen oxidase inhibitors. The tolerance trait has beenfound to be closely linked to a number of molecular markers that map tolinkage groups L and N. Soybean plants, seeds, tissue cultures, variantsand mutants having tolerance to protoporphyrinogen oxidase inhibitorsproduced by the foregoing methods are also provided in this invention.

The QTL associated with tolerance to protoporphyrinogen oxidaseinhibitors maps to soybean linkage group L and/or N. These QTL may bemapped by one or more molecular markers. For linkage group L, themarkers include SATT495, P10649C-3, SATT182, SATT388, SATT313, SATT613,S08102-1-Q1, S08103-1-Q1. S08104-1-Q1, S08106-1-Q1, S08107-1-Q1,S08107-1-Q1, S08109-1-Q1, S08110-1-Q1, S08111-1-Q1, S08115-2-Q1,S08117-1-Q1, S08119-1-Q1, S08116-1-Q1, S08112-1-Q1, S08108-1-Q1,S08101-4-Q1, S08101-1-Q1, S08101-2-Q1, and S08101-3-Q1, or markersclosely linked thereto. Other markers of linkage group L may also beused to identify the presence or absence of the gene, including othermarkers above marker SATT613. For linkage group N, the markers includeSat_(—)379, SCT_(—)195, SATT631, S60167-TB, SATT675, SATT624, SATT080,SATT387, or markers closely linked thereto. Other markers of linkagegroup N may also be used to identify the presence or absence of thegene, including other markers above marker SATT387.

The information disclosed herein regarding the QTL for tolerance toprotoporphyrinogen oxidase inhibitors which maps to soybean linkagegroup L and/or N is used to aid in the selection of breeding plants,lines and populations containing tolerance to protoporphyrinogen oxidaseinhibitors for use in introgression of this trait into elite soybeangermplasm, or germplasm of proven genetic superiority suitable forvariety release.

Also provided is a method for introgressing a soybean QTL associatedwith tolerance to protoporphyrinogen oxidase inhibitors intonon-tolerant soybean germplasm or less tolerant soybean germplasm.According to the method, nucleic acid markers mapping the QTL are usedto select soybean plants containing the QTL. Plants so selected have ahigh probability of expressing the trait tolerance to protoporphyrinogenoxidase inhibitors. Plants so selected can be used in a soybean breedingprogram. Through the process of introgression, the QTL associated withtolerance to protoporphyrinogen oxidase inhibitors is introduced fromplants identified using marker-assisted selection to other plants.According to the method, agronomically desirable plants and seeds can beproduced containing the QTL associated with tolerance toprotoporphyrinogen oxidase inhibitors from germplasm containing the QTL.Sources of tolerance to protoporphyrinogen oxidase inhibitors aredisclosed below.

Also provided herein is a method for producing a soybean plant adaptedfor conferring tolerance to protoporphyrinogen oxidase inhibitors.First, donor soybean plants for a parental line containing the toleranceQTL are selected. According to the method, selection can be accomplishedvia nucleic acid marker-associated selection as explained herein.Selected plant material may represent, among others, an inbred line, ahybrid, a heterogeneous population of soybean plants, or simply anindividual plant. According to techniques well known in the art of plantbreeding, this donor parental line is crossed with a second parentalline. Typically, the second parental line is a high yielding line. Thiscross produces a segregating plant population composed of geneticallyheterogeneous plants. Plants of the segregating plant population arescreened for the tolerance QTL and are subjected to further breeding.This further breeding may include, among other techniques, additionalcrosses with other lines, hybrids, backcrossing, or self-crossing. Theresult is a line of soybean plants that is tolerant toprotoporphyrinogen oxidase inhibitors and also has other desirabletraits from one or more other soybean lines.

Also provided is a method for introgressing a soybean QTL associatedwith tolerance or sensitivity to protoporphyrinogen oxidase inhibitorsinto non-tolerant soybean germplasm or less tolerant soybean germplasm.According to the method, nucleic acid markers mapping the QTL are usedto select soybean plants containing the QTL. Plants so selected have ahigh probability of expressing the trait tolerance or sensitivity toprotoporphyrinogen oxidase inhibitors. Plants so selected can be used ina soybean breeding program. Through the process of introgression, theQTL associated with tolerance or sensitivity to protoporphyrinogenoxidase inhibitors is introduced from plants identified usingmarker-assisted selection to other plants. According to the method,agronomically desirable plants and seeds can be produced containing theQTL associated with tolerance or sensitivity to protoporphyrinogenoxidase inhibitors from germplasm containing the QTL. Sources oftolerance or sensitivity to protoporphyrinogen oxidase inhibitors aredisclosed below.

Also provided herein is a method for producing a soybean plant adaptedfor conferring tolerance or sensitivity to protoporphyrinogen oxidaseinhibitors. First, donor soybean plants for a parental line containingthe tolerance QTL are selected. According to the method, selection canbe accomplished via nucleic acid marker-associated selection asexplained herein. Selected plant material may represent, among others,an inbred line, a hybrid, a heterogeneous population of soybean plants,or simply an individual plant. According to techniques well known in theart of plant breeding, this donor parental line is crossed with a secondparental line. Typically, the second parental line is a high yieldingline. This cross produces a segregating plant population composed ofgenetically heterogeneous plants. Plants of the segregating plantpopulation are screened for the tolerance QTL and are subjected tofurther breeding. This further breeding may include, among othertechniques, additional crosses with other lines, hybrids, backcrossing,or self-crossing. The result is a line of soybean plants that istolerant to mesotrione and/or isoxaflutole herbicides, and also hasother desirable traits, such as yield, from one or more other soybeanlines.

Also described are isolated polynucleotides and isolated polypeptidesrelevant to tolerance or sensitivity to protoporphyrinogen oxidaseinhibitors. Additional traits may also be added to plants having suchtolerance or sensitivity, such as additional herbicide tolerance traits,insect tolerance traits, or other transgenic traits. Also described aremethods of introgressing a tolerance or susceptibility allele into aplant, such as by crossing a soybean plant tolerant to an isoflutoleherbicide with a soybean plant susceptible to a isoflutole herbicide inorder to form a segregating population, screening the segregatingpopulation with one or more nucleic acid markers to determine if plantsfrom the segregating population contains at least one SNP selected fromthe group consisting of an SNP at position #1433, #1559, #1750, #1832,#1932, #2727, #2858, #3027, #3088, #3090, and #3334 of the sequence setforth as SEQ ID NO: 114 as shown in FIG. 3, or a sequence equivalent toSEQ ID NO: 114, and optionally selecting, if present, one or moresoybean plants of the segregating population containing the at least oneSNP. Alternatively, such tolerance may be transgenically provided byintroducing into a plant cell a polynucleotide as disclosed hereinoperably linked to a promoter functional in the plant cell to produce atransformed plant cell, and optionally selecting a transformed plantcell having the polynucleotide stably incorporated into its genome.

Compositions include isolated polynucleotides encoding ABC transporterpolypeptides that confer tolerance to such herbicides, and isolated ABCtransporter polypeptides. Compositions include those polynucleotidesencoding polypeptides with amino acid substitutions at position V520X,L584X, S611X, K953X, L1030X, and/or G1112X or positions equivalentthereto, as well as polypeptides with amino acid substitutions atposition V520X, L584X, S611X, K953X, L1030X, and/or G1112X or positionsequivalent thereto. Also useful are isolated polynucleotide variants,polynucleotides encoding polypeptide variants, and polypeptide variantshaving sequence identity to the appropriate reference sequence, such anABC transporter polypeptide of at least 60%, 65%, 70%, 75, 80%, 81%,82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99%, 99.5%, or 99.75%.

Soybean plants, seeds, tissue cultures, variants and mutants havingtolerance or sensitivity to PPO inhibitor herbicides produced by theforegoing methods are also provided. Also provided herein are methodsfor controlling weeds in a crop by applying to the crop and any weedsaffecting such crop an effective amount of such herbicide(s), eitherpre-emergent or post-emergent, such that the weeds are substantiallycontrolled without substantially negatively impacting the crop.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention can be more fully understood from the following detaileddescription and the accompanying drawings and Sequence Listing, whichform a part of this application.

FIG. 1 Panel A provides an integrated genetic map of soybean markers onlinkage group L, including the marker type (SSR or ASH/SNP). The geneticmap positions of the markers are indicated in centiMorgans (cM),typically with position zero being the first (most distal) marker on thechromosome. The map includes relative positions for some markers forwhich higher resolution genetic mapping data was not available; noposition in cM is provided. Panel B provides a table listing geneticmarkers that are linked to the protoporphyrinogen oxidase (PPOase)inhibitor tolerance markers identified on linkage group L. These markersare from the soybean public composite map of Jun. 18, 2008 for linkagegroup L.

FIG. 2 Panel A provides an integrated genetic map of soybean markers onlinkage group N, including the marker type (SSR or ASH/SNP). The geneticmap positions of the markers are indicated in centiMorgans (cM),typically with position zero being the first (most distal) marker on thechromosome. Panel B provides a table listing genetic markers that arelinked to the protoporphyrinogen oxidase (PPOase) inhibitor tolerancemarkers identified by the present invention on linkage group N. Thesemarkers are from the soybean public composite map of Jun. 18, 2008 forlinkage group N.

FIG. 3 provides a table listing SSR markers, including those markersthat demonstrated linkage disequilibrium with the protoporphyrinogenoxidase (PPOase) inhibitor tolerance phenotype. The table provides thesequences of the left and right PCR primers used in the SSR marker locusgenotyping analysis. Also shown is the pigtail sequence used on the 5′end of the right primer.

FIG. 4 provides a table listing the SNP markers that demonstratedlinkage disequilibrium with the protoporphyrinogen oxidase (PPOase)inhibitor tolerance phenotype. The table provides the sequences of thePCR primers used to generate a SNP-containing amplicon, and theallele-specific probes that were used to identify the SNP allele in anallele-specific hybridization assay (ASH assay).

FIG. 5 provides an example of cultivars with vastly differentprotoporphyrinogen oxidase (PPOase) inhibitor tolerance phenotypes.Shown are field samples, with a non-tolerant variety on the left (whitecircle: stunted, necrotic) and tolerant variety on the right (normalgrowth)

FIG. 6 provides an example of cultivars with vastly differentprotoporphyrinogen oxidase (PPOase) inhibitor tolerance phenotypes.Shown are greenhouse samples, with a non-tolerant variety withnon-tolerant (arrow, left side) and tolerant (right side) varietychecks, showing treated plants in the foreground, and untreated plantsin the background.

DETAILED DESCRIPTION

It is to be understood that this invention is not limited to particularembodiments or examples, which can, of course, vary. It is also to beunderstood that the terminology used herein is for the purpose ofdescribing particular embodiments only, and is not intended to belimiting. As used in this specification and the appended claims, termsin the singular and the singular forms “a,” “an” and “the,” for example,include plural referents unless the content clearly dictates otherwise.Thus, for example, reference to “plant,” “the plant” or “a plant” alsoincludes a plurality of plants; also, depending on the context, use ofthe term “plant” can also include genetically similar or identicalprogeny of that plant; use of the term “a nucleic acid” optionallyincludes, as a practical matter, many copies of that nucleic acidmolecule; similarly, the term “probe” optionally (and typically)encompasses many similar or identical probe molecules.

Certain definitions used in the specification are provided below. Alsoin the examples which follow, a number of terms are used. Terms notspecifically defined herein should be given their ordinary meaning tothose in the art. In order to provide a clear and consistentunderstanding of the specification and claims, including the scope to begiven such terms, the following definitions are provided:

AGRONOMICS, AGRONOMIC TRAITS, and AGRONOMIC PERFORMANCE refer to thetraits and underlying genetic elements of a given plant variety thatcontribute to yield over the course of growing season. Individualagronomic traits include emergence vigor, vegetative vigor, stresstolerance, disease resistance or tolerance, herbicide resistance ortolerance, branching, flowering, seed set, seed size, seed density,standability, threshability and the like.

ALLELE means any of one or more alternative forms of a genetic sequence.In a diploid cell or organism, the two alleles of a given sequencetypically occupy corresponding loci on a pair of homologous chromosomes.

The term AMPLIFYING in the context of nucleic acid amplification is anyprocess whereby additional copies of a selected nucleic acid (or atranscribed form thereof) are produced. Typical amplification methodsinclude various polymerase based replication methods, including thepolymerase chain reaction (PCR), ligase mediated methods such as theligase chain reaction (LCR) and RNA polymerase based amplification(e.g., by transcription) methods. An “amplicon” is an amplified nucleicacid, e.g., a nucleic acid that is produced by amplifying a templatenucleic acid by any available amplification method (e.g., PCR, LCR,transcription, or the like).

An ANCESTRAL LINE is a parent line used as a source of genes.

An ANCESTRAL POPULATION is a group of ancestors that have contributedthe bulk of the genetic variation that was used to develop elite lines.

BACKCROSSING is a process in which a breeder crosses a progeny varietyback to one of the parental genotypes one or more times.

BREEDING means the genetic manipulation of living organisms.

The term CHROMOSOME SEGMENT designates a contiguous linear span ofgenomic DNA that resides in planta on a single chromosome.

CULTIVAR and VARIETY are used synonymously and mean a group of plantswithin a species (e.g., Glycine max) that share certain genetic traitsthat separate them from the typical form and from other possiblevarieties within that species. Soybean cultivars are inbred linesproduced after several generations of self-pollinations. Individualswithin a soybean cultivar are homogeneous, nearly genetically identical,with most loci in the homozygous state.

An ELITE LINE is an agronomically superior line that has resulted frommany cycles of breeding and selection for superior agronomicperformance. Numerous elite lines are available and known to those ofskill in the art of soybean breeding.

An ELITE POPULATION is an assortment of elite individuals or lines thatcan be used to represent the state of the art in terms of agronomicallysuperior genotypes of a given crop species, such as soybean.

A GENETIC MAP is a description of genetic linkage relationships amongloci on one or more chromosomes or linkage groups within a givenspecies, generally depicted in a diagrammatic or tabular form.

GENOTYPE refers to the genetic constitution of a cell or organism.

GERMPLASM means the genetic material that comprises the physicalfoundation of the hereditary qualities of an organism. As used herein,germplasm includes seeds and living tissue from which new plants may begrown; or, another plant part, such as leaf, stem, pollen, or cells,that may be cultured into a whole plant. Germplasm resources providesources of genetic traits used by plant breeders to improve commercialcultivars.

An individual is HOMOZYGOUS if the individual has only one type ofallele at a given locus (e.g., a diploid individual has a copy of thesame allele at a locus for each of two homologous chromosomes). Anindividual is “HETEROZYGOUS” if more than one allele type is present ata given locus (e.g., a diploid individual with one copy each of twodifferent alleles). The term “HOMOGENEITY” indicates that members of agroup have the same genotype at one or more specific loci. In contrast,the term “HETEROGENEITY” is used to indicate that individuals within thegroup differ in genotype at one or more specific loci.

INTROGRESSION means the entry or introduction of a gene, QTL, or traitlocus from the genome of one plant into the genome of another plant.

A LINE or a STRAIN is a group of individuals of identical parentage thatare generally inbred to some degree and that are generally homozygousand homogeneous at most loci (isogenic or near isogenic). A “SUBLINE”refers to an inbred subset of descendents that are genetically distinctfrom other similarly inbred subsets descended from the same progenitor.Traditionally, a subline has been derived by inbreeding the seed from anindividual soybean plant selected at the F3 to F5 generation until theresidual segregating loci are “fixed” or homozygous across most or allloci. Commercial soybean varieties (or lines) are typically produced byaggregating (“bulking”) the self-pollinated progeny of a single F3 to F5plant from a controlled cross between 2 genetically different parents.While the variety typically appears uniform, the self-pollinatingvariety derived from the selected plant eventually (e.g., F8) becomes amixture of homozygous plants that can vary in genotype at any locus thatwas heterozygous in the originally selected F3 to F5 plant. Marker-basedsublines that differ from each other based on qualitative polymorphismat the DNA level at one or more specific marker loci are derived bygenotyping a sample of seed derived from individual self-pollinatedprogeny derived from a selected F3-F5 plant. The seed sample can begenotyped directly as seed, or as plant tissue grown from such a seedsample. Optionally, seed sharing a common genotype at the specifiedlocus (or loci) are bulked providing a subline that is geneticallyhomogenous at identified loci important for a trait of interest (yield,tolerance, etc.).

LINKAGE refers to a phenomenon wherein alleles on the same chromosometend to segregate together more often than expected by chance if theirtransmission was independent. Genetic recombination occurs with anassumed random frequency over the entire genome. Genetic maps areconstructed by measuring the frequency of recombination between pairs oftraits or markers. The closer the traits or markers lie to each other onthe chromosome, the lower the frequency of recombination, and thegreater the degree of linkage. Traits or markers are considered hereinto be linked if they generally co-segregate. A 1/100 probability ofrecombination per generation is defined as a map distance of 1.0centiMorgan (1.0 cM). For example, in soybean, 1 cM correlates, onaverage, to about 400,000 base pairs (400 Kb).

The genetic elements or genes located on a single chromosome segment arephysically linked. In the context of the present invention the geneticelements located within a chromosome segment are also geneticallylinked, typically within a genetic recombination distance of less thanor equal to 50 centimorgans (cM), e.g., about 49, 40, 30, 20, 10, 5, 4,3, 2, 1, 0.75, 0.5, or 0.25 cM or less. That is, two genetic elementswithin a single chromosome segment undergo recombination during meiosiswith each other at a frequency of less than or equal to about 50%, e.g.,about 49%, 40%, 30%, 20%, 10%, 5%, 4%, 3%, 2%, 1%, 0.75%, 0.5%, or 0.25%or less.

LINKAGE GROUP refers to traits or markers that generally co-segregate. Alinkage group generally corresponds to a chromosomal region containinggenetic material that encodes the traits or markers.

LOCUS is a defined segment of DNA.

A MAP LOCATION is an assigned location on a genetic map relative tolinked genetic markers where a specified marker can be found within agiven species. Markers are frequently described as being “above” or“below” other markers on the same linkage group; a marker is “above”another marker if it appears earlier on the linkage group, whereas amarker is “below” another marker if it appears later on the linkagegroup.

MAPPING is the process of defining the linkage relationships of locithrough the use of genetic markers, populations segregating for themarkers, and standard genetic principles of recombination frequency.

MOLECULAR MARKER is a nucleic acid or amino acid sequence that issufficiently unique to characterize a specific locus on the genome.Examples include Restriction Fragment Length Polymorphisms (RFLPs),Single Sequence Repeats (SSRs), Target Region AmplificationPolymorphisms (TRAPs), Isozyme Electrophoresis, Randomly AmplifiedPolymorphic DNAs (RAPDs), Arbitrarily Primed Polymerase Chain Reaction(AP-PCR), DNA Amplification Fingerprinting (DAF), Sequence CharacterizedAmplified Regions (SCARs), Amplified Fragment Length Polymorphisms(AFLPs), and Single Nucleotide Polymorphisms (SNPs). Additionally, othertypes of molecular markers are known to the art, and phenotypic traitsmay also be used as markers in the methods. All markers are used todefine a specific locus on the soybean genome. Large numbers of thesemarkers have been mapped. Each marker is therefore an indicator of aspecific segment of DNA, having a unique nucleotide sequence. The mappositions provide a measure of the relative positions of particularmarkers with respect to one another. When a trait is stated to be linkedto a given marker it will be understood that the actual DNA segmentwhose sequence affects the trait generally co-segregates with themarker. More precise and definite localization of a trait can beobtained if markers are identified on both sides of the trait. Bymeasuring the appearance of the marker(s) in progeny of crosses, theexistence of the trait can be detected by relatively simple moleculartests without actually evaluating the appearance of the trait itself,which can be difficult and time-consuming because the actual evaluationof the trait requires growing plants to a stage where the trait can beexpressed. Molecular markers have been widely used to determine geneticcomposition in soybeans. Shoemaker and Olsen, ((1993) Molecular LinkageMap of Soybean (Glycine max L. Merr.). p. 6.131-6.138. In S. J. O'Brien(ed.) Genetic Maps: Locus Maps of Complex Genomes. Cold Spring HarborLaboratory Press. Cold Spring Harbor, N.Y.), developed a moleculargenetic linkage map that consisted of 25 linkage groups with about 365RFLP, 11 RAPD (random amplified polymorphic DNA), three classicalmarkers, and four isozyme loci. See also Shoemaker R. C. 1994 RFLP Mapof Soybean. P. 299-309 In R. L. Phillips and I. K. Vasil (ed.) DNA-basedmarkers in plants. Kluwer Academic Press Dordrecht, the Netherlands.

MARKER ASSISTED SELECTION refers to the process of selecting a desiredtrait or desired traits in a plant or plants by detecting one or moremolecular markers from the plant, where the molecular marker is linkedto the desired trait.

The term PHYSICALLY LINKED is used to indicate that two loci, e.g., twomarker loci, are physically present on the same chromosome.Advantageously, the two loci are located in close proximity such thatrecombination between homologous chromosome pairs does not occur betweenthe two loci during meiosis with high frequency, e.g., such that linkedloci co-segregate at least about 90% of the time, e.g., 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.75%, or more of the time.

The term PLANT includes reference to an immature or mature whole plant,including a plant from which seed or grain or anthers have been removed.Seed or embryo that will produce the plant is also considered to be theplant.

PLANT PARTS include leaves, stems, buds, roots, root tips, anthers,seed, grain, embryo, pollen, ovules, flowers, cotyledons, hypocotyls,pods, flowers, shoots and stalks, tissues, cells and the like.

POLYMORPHISM means a change or difference between two related nucleicacids. A “NUCLEOTIDE POLYMORPHISM” refers to a nucleotide that isdifferent in one sequence when compared to a related sequence when thetwo nucleic acids are aligned for maximal correspondence. A “GENETICNUCLEOTIDE POLYMORPHISM” refers to a nucleotide that is different in onesequence when compared to a related sequence when the two nucleic acidsare aligned for maximal correspondence, where the two nucleic acids aregenetically related, i.e., homologous, for example, where the nucleicacids are isolated from different strains of a soybean plant, or fromdifferent alleles of a single strain, or the like.

PROBE means a polynucleotide designed to be sufficiently complementaryto a sequence in a denatured nucleic acid to be probed and to be boundunder selected stringency conditions.

RAPD marker means random amplified polymorphic DNA marker. Chance pairsof sites complementary to single octa- or decanucleotides may exist inthe correct orientation and close enough to one another for PCRamplification. With some randomly chosen decanucleotides no sequencesare amplified. With others, the same length products are generated fromDNAs of different individuals. With still others, patterns of bands arenot the same for every individual in a population. The variable bandsare commonly called random amplified polymorphic DNA (RAPD) bands.

RECOMBINATION FREQUENCY is the frequency of a crossing over event(recombination) between two genetic loci. Recombination frequency can beobserved by following the segregation of markers and/or traits duringmeiosis. A marker locus is “associated with” another marker locus orsome other locus (for example, a tolerance locus), when the relevantloci are part of the same linkage group and are in linkagedisequilibrium. This occurs when the marker locus and a linked locus arefound together in progeny plants more frequently than if the two locisegregate randomly. Similarly, a marker locus can also be associatedwith a trait, e.g., a marker locus can be “associated with tolerance orimproved tolerance” when the marker locus is in linkage disequilibriumwith the trait.

RFLP means restriction fragment length polymorphism. Molecular markersthat occur because any sequence change in DNA, including a single basechange, insertion, deletion or inversion, can result in loss or gain ofa restriction endonuclease recognition site. The size and number offragments generated by one such enzyme is therefore altered. A probethat hybridizes specifically to DNA in the region of such an alterationcan be used to rapidly and specifically identify a region of DNA thatdisplays allelic variation between two plant varieties. IsozymeElectrophoresis and RFLPs have been widely used to determine geneticcomposition

SELF CROSSING or SELF-POLLINATION or SELFING is a process through whicha breeder crosses hybrid progeny with itself; for example, a secondgeneration hybrid F2 with itself to yield progeny designated F2:3.

SNP means single nucleotide polymorphism. SNPs are genetic markers inwhich DNA sequence variations that occur when a single nucleotide (A, T,C, or G) in the genome sequence is altered are mapped to sites on thesoybean genome. Many techniques for detecting SNPs are known in the art,including allele specific hybridization, primer extension, and directsequencing.

SSR means short sequence repeats. SSRs are genetic markers based onpolymorphisms in repeated nucleotide sequences, such as microsatellites.A marker system based on SSRs can be highly informative in linkageanalysis relative to other marker systems in that multiple alleles maybe present. The PCR detection is done by use of two oligonucleotideprimers flanking the polymorphic segment of repetitive DNA. Repeatedcycles of heat denaturation of the DNA followed by annealing of theprimers to their complementary sequences at low temperatures, andextension of the annealed primers with DNA polymerase, comprise themajor part of the methodology.

TOLERANT and TOLERANCE refer to plants in which higher doses of aherbicide are required to produce effects similar to those seen innon-tolerant plants. Tolerant plants typically exhibit fewer necrotic,lytic, chlorotic, or other lesions when subjected to the herbicide atconcentrations and rates typically employed by the agriculturalcommunity.

TRANSGENIC PLANT refers to a plant that comprises within its cells aheterologous polynucleotide. Generally, the heterologous polynucleotideis stably integrated within the genome such that the polynucleotide ispassed on to successive generations. The heterologous polynucleotide maybe integrated into the genome alone or as part of a recombinantexpression cassette. TRANSGENIC is used herein to refer to any cell,cell line, callus, tissue, plant part or plant, the genotype of whichhas been altered by the presence of heterologous nucleic acid includingthose transgenic organisms or cells initially so altered, as well asthose created by crosses or asexual propagation from the initialtransgenic organism or cell. The term “transgenic” as used herein doesnot encompass the alteration of the genome (chromosomal orextra-chromosomal) by conventional plant breeding methods (e.g.,crosses) or by naturally occurring events such as randomcross-fertilization, non-recombinant viral infection, non-recombinantbacterial transformation, non-recombinant transposition, or spontaneousmutation.

TRAP marker means target region amplification polymorphism marker. TheTRAP technique employs one fixed primer of known sequence in combinationwith a random primer to amplify genomic fragments. The differences infragments between alleles can be detected by gel electrophoresis.

The term VECTOR is used in reference to polynucleotide or othermolecules that transfer nucleic acid segment(s) into a cell. The term“vehicle” is sometimes used interchangeably with “vector.” A vectoroptionally comprises parts which mediate vector maintenance and enableits intended use (e.g., sequences necessary for replication, genesimparting drug or antibiotic resistance, a multiple cloning site,operably linked promoter/enhancer elements which enable the expressionof a cloned gene, etc.). Vectors are often derived from plasmids,bacteriophages, or plant or animal viruses. A “cloning vector” or“shuttle vector” or “subcloning vector” contains operably linked partsthat facilitate subcloning steps (e.g., a multiple cloning sitecontaining multiple restriction endonuclease sites).

The term YIELD refers to the productivity per unit area of a particularplant product of commercial value. For example, yield of soybean iscommonly measured in bushels of seed per acre or metric tons of seed perhectare per season. Yield is affected by both genetic and environmentalfactors. Yield is the final culmination of all agronomic traits.

An equivalent position in a polynucleotide and/or polypeptide sequenceis a position that correlates a position in the reference sequence whenthe sequences are aligned for a maximum correspondence. In some examplesthe sequences are aligned across their whole length using a globalalignment program. In other examples, a portion of the sequence orsequences may be aligned using a local alignment program or a globalalignment program, for example a sequence may comprise exons andintrons, conserved motifs or domains, or functional motifs or domainswhich may be aligned to the reference sequence(s) to identify equivalentpositions. Equivalent positions in polynucleotides encoding apolypeptide can be determined using the encoded amino acid, and/or usinga FrameAlign program to align the polynucleotide and polypeptide formaximal correspondence.

The term “homologous” refers to nucleic acid sequences that are derivedfrom a common ancestral gene through natural or artificial processes(e.g., are members of the same gene family), and thus, typically sharesequence similarity. Typically, homologous nucleic acids have sufficientsequence identity that one of the sequences or a subsequence thereof orits complement is able to selectively hybridize to the other underselective (e.g., stringent) hybridization conditions. The term“selectively hybridizes” includes reference to hybridization, understringent hybridization conditions, of a nucleic acid sequence to aspecified nucleic acid target sequence to a detectably greater degree(e.g., at least 2-fold over background) than its hybridization tonon-target nucleic acid sequences and to the substantial exclusion ofnon-target nucleic acids. Selectively hybridizing nucleic acid sequencestypically have about at least 70% sequence identity, at least 80%sequence identity, or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, 99.5%, 99.75%, or 100% sequence identity with each other. A nucleicacid that exhibits at least some degree of homology to a referencenucleic acid can be unique or identical to the reference nucleic acid orits complementary sequence.

The term “isolated” refers to material, such as polynucleotides orpolypeptides, which are identified and separated from at least onecontaminant with which it is ordinarily associated in its natural ororiginal source. Furthermore, an isolated polynucleotide or polypeptideis typically present in a form or setting that is different from theform or setting that is normally found in nature. In some examples, theisolated molecule is substantially free from components that normallyaccompany or interact with it in its naturally occurring environment. Insome embodiments, the isolated material optionally comprises materialnot found with the material in its natural environment, e.g., in a cell.

As used herein, the terms “exogenous” or “heterologous” as applied topolynucleotides or polypeptides refers to molecules that have beenartificially supplied to a biological system (e.g., a plant cell, aplant gene, a particular plant species or a plant chromosome understudy) and are not native to that particular biological system. Theterms indicate that the relevant material originated from a source otherthan the naturally occurring source, or refers to molecules having anon-natural configuration, genetic location or arrangement of parts. Aheterologous polynucleotide includes polynucleotides from anotherorganism or the same organism which have been modified by linkage to adistinct non-endogenous polynucleotide and/or inserted to a distinctnon-endogenous locus. The terms “exogenous” and “heterologous” aresometimes used interchangeably with “recombinant.”

In contrast, for example, a “native” or “endogenous” gene is a gene thatdoes not contain nucleic acid elements encoded by sources other than thechromosome or other genetic element on which it is normally found innature. An endogenous gene, transcript or polypeptide is encoded by itsnatural chromosomal locus, and not artificially supplied to the cell.

The term “recombinant” indicates that the material (e.g., a recombinantnucleic acid, gene, polynucleotide or polypeptide) has been altered byhuman intervention. Generally, the arrangement of parts of a recombinantmolecule is not a native configuration, or the primary sequence of therecombinant polynucleotide or polypeptide has in some way beenmanipulated. The alteration to yield the recombinant material can beperformed on the material within or removed from its natural environmentor state. For example, a naturally occurring nucleic acid becomes arecombinant nucleic acid if it is altered, or if it is transcribed fromDNA which has been altered, by means of human intervention performedwithin the cell from which it originates. A gene sequence open readingframe is recombinant if that nucleotide sequence has been removed fromit natural text and cloned into any type of artificial nucleic acidvector. Protocols and reagents to produce recombinant molecules,especially recombinant nucleic acids, are common and routine in the art(see, e.g., Maniatis et al. (eds.), Molecular Cloning: A LaboratoryManual, Cold Spring Harbor Laboratory Press, NY, [1982]; Sambrook et al.(eds.), Molecular Cloning: A Laboratory Manual, Second Edition, Volumes1-3, Cold Spring Harbor Laboratory Press, NY, [1989]; and Ausubel et al.(eds.), Current Protocols in Molecular Biology, Vol. 1-4, John Wiley &Sons, Inc., New York [1994]). The term recombinant can also refer to anorganism that harbors a recombinant material, e.g., a plant thatcomprises a recombinant nucleic acid is considered a recombinant plant.In some embodiments, a recombinant organism is a transgenic organism.

The term “introduced” when referring to a heterologous or exogenousnucleic acid refers to the incorporation of a nucleic acid into aeukaryotic or prokaryotic cell using any type of suitable vector, e.g.,naked linear DNA, plasmid, plastid or virion), converted into anautonomous replicon, or transiently expressed (e.g., transfected mRNA).The term includes such nucleic acid introduction means as“transfection,” “transformation” and “transduction.”

The term “host cell” means a cell that contains a heterologous nucleicacid, such as a vector, and supports the replication and/or expressionof the nucleic acid. Host cells may be prokaryotic cells such as E.coli, or eukaryotic cells such as yeast, insect, amphibian or mammaliancells. In some examples, host cells are plant cells, including but notlimited to dicot and monocot cells.

The term “transgenic plant” refers to a plant that comprises within itscells a heterologous polynucleotide. Generally, the heterologouspolynucleotide is stably integrated within the genome such that thepolynucleotide is passed on to successive generations. The heterologouspolynucleotide may be integrated into the genome alone or as part of arecombinant expression cassette. “Transgenic” is used herein to refer toany cell, cell line, callus, tissue, plant part or plant, the genotypeof which has been altered by the presence of heterologous nucleic acidincluding those transgenic organisms or cells initially so altered, aswell as those created by crosses or asexual propagation from the initialtransgenic organism or cell. The term “transgenic” as used herein doesnot encompass the alteration of the genome (chromosomal orextra-chromosomal) by conventional plant breeding methods (e.g.,crosses) or by naturally occurring events such as randomcross-fertilization, non-recombinant viral infection, non-recombinantbacterial transformation, non-recombinant transposition, or spontaneousmutation.

Plant cell, as used herein includes, without limitation, cells within orderived from, for example and without limitation, plant seeds, planttissue suspension cultures, plant tissue, plant tissue explants, plantembryos, meristematic tissue, callus tissue, leaves, roots, shoots,gametophytes, sporophytes, pollen and microspores.

The term “crossed” or “cross” means the fusion of gametes viapollination to produce progeny (e.g., cells, seeds or plants). The termencompasses both sexual crosses (the pollination of one plant byanother) and selfing (self-pollination, e.g., when the pollen and ovuleare from the same plant).

The term “introgression” refers to the transmission of a desired alleleof a genetic locus from one genetic background to another. For example,introgression of a desired allele at a specified locus can betransmitted to at least one progeny plant via a sexual cross between twoparent plants, at least one of the parent plants having the desiredallele within its genome. Alternatively, for example, transmission of anallele can occur by recombination between two donor genomes, e.g., in afused protoplast, where at least one of the donor protoplasts has thedesired allele in its genome. The desired allele can be, e.g., atransgene or a gene allele that imparts resistance to a plant pathogen.

Protoporphyrinogen Oxidase Inhibitors

Porphyrins are biologically important organic structures that are foundin plants attached to chlorophyll and cytochrome pigments. Anintermediate in the chlorophyll and cytochrome synthesis pathway isprotoporphyrinogen IX which is converted to protoporphyrin IX byprotoporphyrinogen oxidase Inhibition of protoporphyrinogen oxidaseprevents this conversion and results in a buildup of protoporphyrinogenIX in the cytoplasm of the plant. The protoporphyrinogen then undergoesnon-enzymatic auto-oxidation and becomes protoporphyrin IX. Whencytoplasmic protoporphyrin IX is exposed to sunlight, free radicals areformed which results in lipid peroxidation reactions that result inplant death. Protoporphyrinogen oxidase inhibitor chemical familiesinclude diphenyl ether, triazolinone, N-phenylphthalimide,pyrimidindione and oxadiazole families. There are other families ofchemistries that also belong to this group.

The diphenyl ether family is characterized by two benzene rings linkedwith an ether bridge and a nitro group bonded to the 4 position.Examples of diphenyl ether protoporphyrinogen oxidase inhibitors includeacifluorfen, fomesafen, oxyfluorfen and lactofen. The diphenyl ethersare typically considered to be contact herbicides.

The triazolinone family is characterized by a 5-member ring containingthree nitrogen atoms (two of which are adjacent) and two carbon atoms,one of the carbon atoms has a double bond with an oxygen atom and one ofthe nitrogen atoms is bonded to a benzene ring. Examples of triazolinoneprotoprophyrinogen oxidase inhibitors include sulfentrazone,carfentrasone, and azafeniden.

The N-phenylphthalimide family is characterized by phthalimide groupwherein the nitrogen is bonded to a benzene ring. Examples ofN-phenylphthalimide protoporphyrinogen oxidase inhibitors includeflumiclorac and flumioxazin.

The oxadiazole family is characterized by a five member ring consistingof two adjacent nitrogen atoms, two carbon atoms, and an oxygen orsulfur atom. Examples of oxadiazole protoporphyrinogen oxidaseinhibitors include oxadiazon and fluthiacet.

The various families of protoporphyrinogen oxidase inhibitors provide awide variety in application options. Sulfentrazone, for example, has arelatively long half-life (approximately 280 days), is known to haveresidual soil activity and is frequently used as a pre-emergenceherbicide. Carfentrazone has a considerably shorter half-life(approximately 4 days) has no residual soil activity, and is used as acontact/post-emergence herbicide. The pyrimidindiones family of PPOherbicides is a rather small class that includes benzfendizone,butagenacil and saflufenacil. This diversity in chemical characteristicscombined with protoporphyrinogen oxidase inhibitor tolerance providesfarmers with a wide variety of weed management options.

Molecular Markers and Genetic Linkage

Molecular markers have been used to selectively improve soybean cropsthrough the use of marker assisted selection. Any detectable polymorphictrait can be used as a marker so long as it is inherited differentiallyand exhibits linkage disequilibrium with a phenotypic trait of interest.A number of soybean markers have been mapped and linkage groups created,as described in Cregan, P. B. et al., “An Integrated Genetic Linkage Mapof the Soybean Genome” (1999) Crop Science 39:1464-90, and more recentlyin Choi et al., “A Soybean Transcript Map: Gene Distribution, Haplotypeand Single-Nucleotide Polymorphism Analysis” (2007) Genetics 176:685-96.Many soybean markers are publicly available at the USDA affiliatedsoybase website.

Most plant traits of agronomic importance are polygenic, otherwise knownas quantitative, traits. A quantitative trait is controlled by severalgenes located at various locations, or loci, in the plant's genome. Themultiple genes have a cumulative effect which contributes to thecontinuous range of phenotypes observed in many plant traits. Thesegenes are referred to as quantitative trait loci (QTL). Recombinationfrequency measures the extent to which a molecular marker is linked witha QTL. Lower recombination frequencies, typically measured incentiMorgans (cM), indicates greater the linkage between the QTL and themolecular marker. The extent to which two features are linked is oftenreferred to as the genetic distance. The genetic distance is alsotypically related to the physical distance between the marker and theQTL, however, certain biological phenomenon (including recombinational“hot spots”) can affect the relationship between physical distance andgenetic distance. Generally, the usefulness of a molecular marker isdetermined by the genetic and physical distance between the marker andthe selectable trait of interest.

The method for determining the presence or absence of a QTL associatedwith tolerance to protoporphyrinogen oxidase inhibitors in soybeangermplasm, comprises analyzing genomic DNA from a soybean germplasm forthe presence of at least one molecular marker, wherein at least onemolecular marker is linked to the QTL, and wherein the QTL maps tosoybean major linkage group L and N and is associated with tolerance toprotoporphyrinogen oxidase inhibitors. The term “is associated with” inthis context means that the QTL associated with tolerance toprotoporphyrinogen oxidase inhibitors has been found, usingmarker-assisted analysis, to be present in soybean plants showingtolerance to protoporphyrinogen oxidase inhibitors in live bioassays asdescribed herein.

Generally, markers that map closer to the QTL mapped to linkage group Land N and associated with tolerance to protoporphyrinogen oxidaseinhibitors are superior to markers that map farther from the QTL. Insome examples a marker used to determine the presence or absence of aQTL mapping to soybean linkage group L and/or N and associated withtolerance to protoporphyrinogen oxidase inhibitors maps to soybeanlinkage group L are SATT495, P10649C-3, SATT182, SATT388, SATT313,SATT613 (or other markers above marker SATT613), S08102-1-Q1,S08103-1-Q1. S08104-1-Q1, S08106-1-Q1, S08107-1-Q1, S08107-1-Q1,S08109-1-Q1, S08110-1-Q1, S08111-1-Q1, S08115-2-Q1, S08117-1-Q1,S08119-1-Q1, S08116-1-Q1, S08112-1-Q1, S08108-1-Q1, S08101-4-Q1,S08101-1-Q1, S08101-2-Q1, and S08101-3-Q1, and those mapped to linkagegroup N are Sat_(—)379, SCT_(—)195, SATT631, S60167-TB, SATT675,SATT624, SATT080, and SATT387 (or other markers above SATT387). Anymarker assigned to soybean linkage group L and/or N and linked to amarker disclosed herein as associated with tolerance toprotoporphyrinogen oxidase inhibitors may be used with the invention.Generally, a linked marker is within 50 cM of the referenced marker.Updated information regarding markers assigned to soybean linkage groupL and N may be found on the USDA's Soybase website. Further, linkagegroup L is now formally referred to as chromosome #19 and linkage groupN is now formally referred to as chromosome #3.

Markers flanking the QTL associated with tolerance to protoporphyrinogenoxidase inhibitors are used in the marker-assisted selection processesprovided. The genomic DNA of soybean germplasm is typically tested forthe presence of at least two of the foregoing molecular markers, onemarker on each side of the QTL. In some examples a QTL on linkage groupL is used. Useful markers on linkage group L include SATT495, P10649C-3,SATT182, SATT388, SATT313, and SATT613, including markers above SATT613.Markers that map close to SATT495, P10649C-3, SATT182, SATT388, SATT313,and SATT613 can also be used. In some examples a QTL on linkage group Nis used. Useful markers on linkage group N include Sat_(—)379,SCT_(—)195, SATT631, S60167-TB, SATT675, SATT624, SATT080, and SATT387,including markers above SATT387. Markers that map close to Sat_(—)379,SCT_(—)195, SATT631, S60167-TB, SATT675, SATT624, SATT080, and SATT387can also be used.

Fine mapping further isolated the location of the QTL to a 56 kbinterval between marker S08117-1-Q1 and S08105-1-Q1 on linkage group L.Accordingly, markers that map within the interval defined by andincluding these markers are particularly useful for selecting for thisQTL. These markers include S08117-1-Q1, S08119-1-Q1, S08118-1-Q1,S08116-1-Q1, S08101-1-Q1, S08112-1-Q1, S08108-1-Q1, S08101-1-Q1,S08101-2-Q1, S08101-3-Q1, S08101-4-Q1, and S08105-4-Q1.

Methods of introgressing protoporphyrinogen oxidase inhibitor toleranceinto non-tolerant or less-tolerant soybean germplasm are provided. Anymethod for introgressing QTLs into soybean plants can be used. In someexamples, a first soybean germplasm that contains tolerance toprotoporphyrinogen oxidase inhibitors derived from the QTL mapped tolinkage group L and/or N which is associated with tolerance toprotoporphyrinogen oxidase inhibitors and a second soybean germplasmthat lacks tolerance to protoporphyrinogen oxidase inhibitors derivedfrom the QTL mapped to linkage group L and/or N are provided. The firstsoybean plant may be crossed with the second soybean plant to provideprogeny soybeans. Phenotypic and/or marker screening is then performedon the progeny plants to determine the presence of tolerance toprotoporphyrinogen oxidase inhibitors derived from the QTL mapped tolinkage group L and/or N. Progeny that test positive for the presence oftolerance to protoporphyrinogen oxidase inhibitors derived from the QTLmapped to linkage group L and/or N can be selected.

In some examples, the screening and selection are performed by usingmarker-assisted selection using any marker or combination of markers onmajor linkage group L and/or N provided. Any method of identifying thepresence or absence of these markers may be used, including for examplesingle-strand conformation polymorphism (SSCP) analysis, base excisionsequence scanning (BESS), RFLP analysis, heteroduplex analysis,denaturing gradient gel electrophoresis, and temperature gradientelectrophoresis, allelic PCR, ligase chain reaction direct sequencing,mini sequencing, nucleic acid hybridization, or micro-array-typedetection.

Systems, including automated systems for selecting plants that comprisea marker of interest and/or for correlating presence of the marker withtolerance are also provided. These systems can include probes relevantto marker locus detection, detectors for detecting labels on the probes,appropriate fluid handling elements and temperature controllers that mixprobes and templates and/or amplify templates, and systems instructionsthat correlate label detection to the presence of a particular markerlocus or allele.

Kits are also provided. For example, a kit can include appropriateprimers or probes for detecting tolerance associated marker loci andinstructions in using the primers or probes for detecting the markerloci and correlating the loci with predicted protoporphyrinogen oxidaseinhibitor tolerance. The kits can further include packaging materialsfor packaging the probes, primers or instructions, controls such ascontrol amplification reactions that include probes, primers or templatenucleic acids for amplifications, molecular size markers, or the like.

Isolated nucleic acid fragments comprising a nucleic acid sequencecoding for soybean tolerance to protoporphyrinogen oxidase inhibitors,are provided. The nucleic acid fragment comprises at least a portion ofnucleic acid belonging to linkage group L and/or N. The nucleic acidfragment is capable of hybridizing under stringent conditions to nucleicacid of a soybean cultivar tolerant to protoporphyrinogen oxidaseinhibitors containing a QTL associated with protoporphyrinogen oxidaseinhibitor tolerance that is located on major linkage group L and/or N.

Vectors comprising such nucleic acid fragments, expression products ofsuch vectors expressed in a host compatible therewith, antibodies to theexpression product (both polyclonal and monoclonal), and antisensenucleic acid to the nucleic acid fragment are also provided.

Seed of a soybean produced by crossing a soybean variety havingprotoporphyrinogen oxidase inhibitor tolerance QTL located on majorlinkage group L and/or N in its genome with another soybean variety, andprogeny thereof, are provided.

Tolerance Markers and Favorable Alleles

In traditional linkage analysis, no direct knowledge of the physicalrelationship of genes on a chromosome is required. Mendel's first law isthat factors of pairs of characteristics are segregated, meaning thatalleles of a diploid trait separate into two gametes and then intodifferent offspring. Classical linkage analysis can be thought of as astatistical description of the relative frequencies of cosegregation ofdifferent traits. Linkage analysis, as described previously, is thewell-characterized descriptive framework of how traits are groupedtogether based upon the frequency with which they segregate together.Because chromosomal distance is approximately proportional to thefrequency of crossing over events between traits, there is anapproximate physical distance that correlates with recombinationfrequency.

Marker loci are traits, and can be assessed according to standardlinkage analysis by tracking the marker loci during segregation. Thus,one cM is equal to a 1% chance that a marker locus will be separatedfrom another locus (which can be any other trait, e.g., another markerlocus, or another trait locus that encodes a QTL), due to crossing overin a single generation. The markers herein, e.g., for linkage group L:SATT495, P10649C-3, SATT182, SATT388, SATT313, SATT613 (and othermarkers above SATT613), S08102-1-Q1, S08103-1-Q1. S08104-1-Q1,S08106-1-Q1, S08107-1-Q1, S08107-1-Q1, S08109-1-Q1, S08110-1-Q1,S08111-1-Q1, S08115-2-Q1, S08117-1-Q1, S08119-1-Q1, S08116-1-Q1,S08112-1-Q1, S08108-1-Q1, S08101-4-Q1, S08101-1-Q1, S08101-2-Q1, andS08101-3-Q1, and for linkage group N: Sat_(—)379, SCT_(—)195, SATT631,S60167-TB, SATT675, SATT624, SATT080, and SATT387 (and other markersabove SATT387), have been found to correlate with tolerance or improvedtolerance to protoporphyrinogen oxidase inhibitors in soybean. Thismeans that the markers are sufficiently proximal to a tolerance traitthat they can be used as a predictor for the tolerance trait itself,using, for example, marker assisted selection (MAS). Soybean plants orgermplasm can be selected for markers or marker alleles that positivelycorrelate with tolerance, without actually raising soybean and measuringfor tolerance or improved tolerance (or, contrawise, soybean plants canbe selected against if they possess markers that negatively correlatewith tolerance or improved tolerance. MAS is a powerful shortcut toselecting for desired phenotypes and for introgressing desired traitsinto cultivars of soybean (e.g., introgressing desired traits into elitelines). MAS is easily adapted to high throughput molecular analysismethods that can quickly screen large numbers of plant or germplasmgenetic material for the markers of interest and is much more costeffective than raising and observing plants for visible traits.

Any marker that is linked to a trait of interest (e.g., in the presentcase, a tolerance or improved tolerance trait) can be used as a markerfor that trait. Thus, in addition to the markers described herein,markers linked to the markers itemized herein can also be used topredict the tolerance or improved tolerance trait. Such linked markersare particularly useful when they are sufficiently proximal to a givenmarker so that they display a low recombination frequency with the givenmarker. Markers closely linked to the markers on linkage group L and/orlinkage group N are also provided. Closely linked markers display across over frequency with a given marker of about 10% or less (the givenmarker is within 10 cM of the given marker). Put another way, closelylinked loci co-segregate at least 90% of the time.

Marker loci are especially useful when they are closely linked to targetloci (e.g., QTL for tolerance, or, alternatively, simply other markerloci, such as those identified herein, that are linked to such QTL) forwhich they are being used as markers. A marker more closely linked to atarget locus is a better indicator for the target locus (due to thereduced cross-over frequency between the target locus and the marker).Thus, in one example, closely linked loci such as a marker locus and asecond locus (e.g., a given marker or a QTL) display an inter-locuscross-over frequency of about 10% or less, about 9% or less, about 8% orless, about 7% or less, about 6% or less, about 5% or less, about 4% orless, about 3% or less, or about 2% or less. In some examples, therelevant loci (e.g., a marker locus and a target locus such as a QTL)display a recombination a frequency of about 1% or less, e.g., about0.75% or less, about 0.5% or less, or about 0.25% or less. Thus, theloci are about 10 cM, 9 cM, 8 cM, 7 cM, 6 cM, 5 cM, 4 cM, 3 cM, 2 cM, 1cM, 0.75 cM, 0.5 cM or 0.25 cM or less apart. Put another way, two locithat are localized to the same chromosome, and at such a distance thatrecombination between the two loci occurs at a frequency of no more than10% (e.g., about 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.75%, 0.5%, 0.25%,or less) are said to be proximal to each other.

When referring to the relationship between two genetic elements, such asa genetic element contributing to tolerance and a proximal marker,“coupling” phase linkage indicates the state where the “favorable”allele at the tolerance locus is physically associated on the samechromosome strand as the “favorable” allele of the respective linkedmarker locus. In coupling phase, both favorable alleles are inheritedtogether by progeny that inherit that chromosome strand. In “repulsion”phase linkage, the “favorable” allele at the locus of interest (e.g., aQTL for tolerance) is physically linked with an “unfavorable” allele atthe proximal marker locus, and the two “favorable” alleles are notinherited together (i.e., the two loci are “out of phase” with eachother).

Optionally, one, two, three or more favorable allele(s) are identifiedin, or introgressed into the plant. Many marker alleles can be selectedfor or against during MAS. Plants or germplasm are identified that haveat least one such favorable allele that positively correlates withtolerance or improved tolerance. However, it is useful for exclusionarypurposes during breeding to identify alleles that negatively correlatewith tolerance, to eliminate such plants or germplasm from subsequentrounds of breeding.

The identification of favorable marker alleles is germplasm-specific.The determination of which marker alleles correlate with tolerance (ornon-tolerance) is determined for the particular germplasm under study.One of skill recognizes that methods for identifying the favorablealleles are routine and well known, and furthermore, that theidentification and use of such favorable alleles is well within thescope of the invention.

Amplification primers for amplifying marker loci and suitable markerprobes to detect marker loci or to genotype SNP alleles are provided.Optionally, other sequences to either side of the given primers can beused in place of the given primers, so long as the primers can amplify aregion that includes the allele to be detected. Further, it will beappreciated that the precise probe to be used for detection can vary,e.g., any probe that can identify the region of a marker amplicon to bedetected can be substituted for those examples provided herein. Theconfiguration of the amplification primers and detection probes can, ofcourse, vary. Thus, the invention is not limited to the primers andprobes specifically recited herein.

In some examples the presence of marker loci is directly detected inunamplified genomic DNA by performing a Southern blot on a sample ofgenomic DNA using probes to the marker loci. Procedures for performingSouthern blotting, amplification (PCR, LCR, or the like) and many othernucleic acid detection methods are well established and are taught,e.g., in Sambrook et al., Molecular Cloning—A Laboratory Manual (3ded.), Vol. 1-3, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.,2000 (“Sambrook”); Current Protocols in Molecular Biology, F. M. Ausubelet al., eds., Current Protocols, a joint venture between GreenePublishing Associates, Inc. and John Wiley & Sons, Inc., (supplementedthrough 2002) (“Ausubel”)) and PCR Protocols A Guide to Methods andApplications (Innis et al. eds) Academic Press Inc. San Diego, Calif.(1990) (Innis). Additional details regarding detection of nucleic acidsin plants can also be found, e.g., in Plant Molecular Biology (1993)Croy (ed.) BIOS Scientific Publishers, Inc.

Separate detection probes can also be omitted in amplification/detectionmethods, e.g., by performing a real time amplification reaction thatdetects product formation by modification of the relevant amplificationprimer upon incorporation into a product, incorporation of labelednucleotides into an amplicon, or by monitoring changes in molecularrotation properties of amplicons as compared to unamplified precursors(e.g., by fluorescence polarization).

Typically, molecular markers are detected by any established methodavailable, including, without limitation, allele specific hybridization(ASH) or other methods for detecting single nucleotide polymorphisms(SNP), amplified fragment length polymorphism (AFLP) detection,amplified variable sequence detection, randomly amplified polymorphicDNA (RAPD) detection, restriction fragment length polymorphism (RFLP)detection, self-sustained sequence replication detection, simplesequence repeat (SSR) detection, single-strand conformationpolymorphisms (SSCP) detection, isozyme markers detection, or the like.While the exemplary markers provided in the tables herein are either SSRor SNP (ASH) markers, any of the aforementioned marker types can beemployed to identify chromosome segments encompassing genetic elementthat contribute to superior agronomic performance (e.g., tolerance orimproved tolerance).

In another example, the presence or absence of a molecular marker isdetermined by nucleotide sequencing of the polymorphic marker region.This method is readily adapted to high throughput analysis as are theother methods noted above, e.g., using available high throughputsequencing methods such as sequencing by hybridization.

In general, the majority of genetic markers rely on one or more propertyof nucleic acids for their detection. For example, some techniques fordetecting genetic markers utilize hybridization of a probe nucleic acidto nucleic acids corresponding to the genetic marker (e.g., amplifiednucleic acids produced using genomic soybean DNA as a template).Hybridization formats, including but not limited to solution phase,solid phase, mixed phase, or in situ hybridization assays are useful forallele detection. An extensive guide to the hybridization of nucleicacids is found in Tijssen (1993) Laboratory Techniques in Biochemistryand Molecular Biology—Hybridization with Nucleic Acid Probes Elsevier,New York, as well as in Sambrook, and Ausubel.

For example, markers that comprise restriction fragment lengthpolymorphisms (RFLP) are detected, e.g., by hybridizing a probe which istypically a sub-fragment (or a synthetic oligonucleotide correspondingto a sub-fragment) of the nucleic acid to be detected to restrictiondigested genomic DNA. The restriction enzyme is selected to providerestriction fragments of at least two alternative (or polymorphic)lengths in different individuals or populations. Determining one or morerestriction enzyme that produces informative fragments for each cross isa simple procedure. After separation by length in an appropriate matrix(e.g., agarose, polyacrylamide, etc.) and transfer to a membrane (e.g.,nitrocellulose, nylon, etc.), the labeled probe is hybridized underconditions which result in equilibrium binding of the probe to thetarget followed by removal of excess probe by washing.

Nucleic acid probes to the marker loci can be cloned and/or synthesized.Any suitable label can be used with a probe. Detectable labels suitablefor use with nucleic acid probes include, for example, any compositiondetectable by spectroscopic, radioisotopic, photochemical, biochemical,immunochemical, electrical, optical or chemical means. Useful labelsinclude biotin for staining with labeled streptavidin conjugate,magnetic beads, fluorescent dyes, radiolabels, enzymes, and colorimetriclabels. Other labels include ligands, which bind to antibodies labeledwith fluorophores, chemiluminescent agents, and enzymes. A probe canalso constitute radiolabelled PCR primers that are used to generate aradiolabelled amplicon. Methods and reagents for labeling nucleic acidsand corresponding detection strategies can be found, e.g., in Haugland(1996) Handbook of Fluorescent Probes and Research Chemicals SixthEdition by Molecular Probes, Inc. (Eugene Oreg.); or Haugland (2001)Handbook of Fluorescent Probes and Research Chemicals Eighth Edition byMolecular Probes, Inc. (Eugene Oreg.).

Amplification-Based Detection Methods

PCR, RT-PCR and LCR are in particularly broad use as amplification andamplification-detection methods for amplifying nucleic acids of interest(e.g., those comprising marker loci), facilitating detection of themarkers. Details regarding the use of these and other amplificationmethods can be found in any of a variety of standard texts, including,e.g., Sambrook, Ausubel, Berger and Croy, supra. Many available biologytexts also have extended discussions regarding PCR and relatedamplification methods. Any RNA can be converted into a double strandedDNA suitable for restriction digestion, PCR expansion and sequencingusing reverse transcriptase and a polymerase (“ReverseTranscription-PCR, or “RT-PCR”). See also Ausubel and Sambrook, supra.

Real Time Amplification/Detection Methods

In one aspect, real time PCR or LCR is performed on the amplificationmixtures described herein, e.g., using molecular beacons or TaqMan™probes. A molecular beacon (MB) is an oligonucleotide or peptide nucleicacid (PNA) which, under appropriate hybridization conditions,self-hybridizes to form a stem and loop structure. The MB has a labeland a quencher at the termini of the oligonucleotide or PNA; thus, underconditions that permit intra-molecular hybridization, the label istypically quenched (or at least altered in its fluorescence) by thequencher. Under conditions where the MB does not display intra-molecularhybridization (e.g., when bound to a target nucleic acid, e.g., to aregion of an amplicon during amplification), the MB label is unquenchedand signal is detected. Standard methods of making and using MBs areknown and MBs and reagents are commercially available. See also, e.g.,Leone et al. (1995) “Molecular beacon probes combined with amplificationby NASBA enable homogenous real-time detection of RNA.” Nucleic AcidsRes. 26:2150-2155; Tyagi and Kramer (1996) “Molecular beacons: probesthat fluoresce upon hybridization” Nature Biotechnology 14:303-308; Blokand Kramer (1997) “Amplifiable hybridization probes containing amolecular switch” Mol Cell Probes 11:187-194; Hsuih et al. (1997)“Novel, ligation-dependent PCR assay for detection of hepatitis C inserum” J Clin Microbiol 34:501-507; Kostrikis et al. (1998) “Molecularbeacons: spectral genotyping of human alleles” Science 279:1228-1229;Sokol et al. (1998) “Real time detection of DNA:RNA hybridization inliving cells” Proc. Natl. Acad. Sci. U.S.A. 95:11538-11543; Tyagi et al.(1998) “Multicolor molecular beacons for allele discrimination” NatureBiotechnology 16:49-53; Bonnet et al. (1999) “Thermodynamic basis of thechemical specificity of structured DNA probes” Proc. Natl. Acad. Sci.U.S.A. 96:6171-6176; Fang et al. (1999) “Designing a novel molecularbeacon for surface-immobilized DNA hybridization studies” J. Am. Chem.Soc. 121:2921-2922; Marras et al. (1999) “Multiplex detection ofsingle-nucleotide variation using molecular beacons” Genet. Anal.Biomol. Eng. 14:151-156; and Vet et al. (1999) “Multiplex detection offour pathogenic retroviruses using molecular beacons” Proc. Natl. Acad.Sci. U.S.A. 96:6394-6399. See also, e.g., U.S. Pat. No. 5,925,517 (Jul.20, 1999) to Tyagi et al. entitled “Detectably labeled dual conformationoligonucleotide probes, assays and kits;” U.S. Pat. No. 6,150,097 toTyagi et al. (Nov. 21, 2000) entitled “Nucleic acid detection probeshaving non-FRET fluorescence quenching and kits and assays includingsuch probes” and U.S. Pat. No. 6,037,130 to Tyagi et al. (Mar. 14,2000), entitled “Wavelength-shifting probes and primers and their use inassays and kits.”

PCR detection and quantification using dual-labeled fluorogenicoligonucleotide probes can be done, using for example TaqMan® probes.These probes are composed of short (e.g., 20-25 base)oligodeoxynucleotides that are labeled with two different fluorescentdyes. On the 5′ terminus of each probe is a reporter dye, and on the 3′terminus of each probe a quenching dye is found. The oligonucleotideprobe sequence is complementary to an internal target sequence presentin a PCR amplicon. When the probe is intact, energy transfer occursbetween the two fluorophores and emission from the reporter is quenchedby the quencher by FRET. During the extension phase of PCR, the probe iscleaved by 5′ nuclease activity of the polymerase used in the reaction,thereby releasing the reporter from the oligonucleotide-quencher andproducing an increase in reporter emission intensity. Accordingly,TaqMan® probes are oligonucleotides that have a label and a quencher,where the label is released during amplification by the exonucleaseaction of the polymerase used in amplification. This provides a realtime measure of amplification during synthesis. A variety of TaqMan®reagents are commercially available, e.g., from Applied Biosystems(Division Headquarters in Foster City, Calif.) as well as from a varietyof specialty vendors such as Biosearch Technologies (e.g., black holequencher probes).

Additional Details Regarding Amplified Variable Sequences, SSR, AFLPASH, SNPs and Isozyme Markers

Amplified variable sequences refer to amplified sequences of the plantgenome, which exhibit high nucleic acid residue variability betweenmembers of the same species. All organisms have variable genomicsequences and each organism (with the exception of a clone) has adifferent set of variable sequences. Once identified, the presence ofspecific variable sequence can be used to predict phenotypic traits.Typically, DNA from the plant serves as a template for amplificationwith primers that flank a variable sequence of DNA. The variablesequence is amplified and then sequenced.

Alternatively, self-sustained sequence replication can be used toidentify genetic markers. Self-sustained sequence replication refers toa method of nucleic acid amplification using target nucleic acidsequences which are replicated exponentially in vitro undersubstantially isothermal conditions by using three enzymatic activitiesinvolved in retroviral replication: (1) reverse transcriptase, (2) RnaseH, and (3) a DNA-dependent RNA polymerase (Guatelli et al. (1990) ProcNatl Acad Sci USA 87:1874). By mimicking the retroviral strategy of RNAreplication by means of cDNA intermediates, this reaction accumulatescDNA and RNA copies of the original target.

Amplified fragment length polymorphisms (AFLP) can also be used asgenetic markers (Vos et al. (1995) Nucl Acids Res 23:4407). The phrase“amplified fragment length polymorphism” refers to selected restrictionfragments, which are amplified before or after cleavage by a restrictionendonuclease. The amplification step allows easier detection of specificrestriction fragments. AFLP allows the detection large numbers ofpolymorphic markers and has been used for genetic mapping of plants(Becker et al. (1995) Mol Gen Genet 249:65; and Meksem et al. (1995) MolGen Genet 249:74).

Allele-specific hybridization (ASH) can be used to identify the geneticmarkers. ASH technology is based on the stable annealing of a short,single-stranded, oligonucleotide probe to a completely complementarysingle-strand target nucleic acid. Detection is via an isotopic ornon-isotopic label attached to the probe.

For each polymorphism, two or more different ASH probes are designed tohave identical DNA sequences except at the polymorphic nucleotides. Eachprobe will have exact homology with one allele sequence so that therange of probes can distinguish all the known alternative allelesequences. Each probe is hybridized to the target DNA. With appropriateprobe design and hybridization conditions, a single-base mismatchbetween the probe and target DNA will prevent hybridization. In thismanner, only one of the alternative probes will hybridize to a targetsample that is homozygous or homogenous for an allele. Samples that areheterozygous or heterogeneous for two alleles will hybridize to both oftwo alternative probes.

ASH markers are used as dominant markers where the presence or absenceof only one allele is determined from hybridization or lack ofhybridization by only one probe. The alternative allele may be inferredfrom the lack of hybridization. ASH probe and target molecules areoptionally RNA or DNA; the target molecules are any length ofnucleotides beyond the sequence that is complementary to the probe; theprobe is designed to hybridize with either strand of a DNA target; theprobe ranges in size to conform to variously stringent hybridizationconditions, etc.

PCR allows the target sequence for ASH to be amplified from lowconcentrations of nucleic acid in relatively small volumes. Otherwise,the target sequence from genomic DNA is digested with a restrictionendonuclease and size separated by gel electrophoresis. Hybridizationstypically occur with the target sequence bound to the surface of amembrane or, as described in U.S. Pat. No. 5,468,613, the ASH probesequence may be bound to a membrane. In one example, ASH data aretypically obtained by amplifying nucleic acid fragments (amplicons) fromgenomic DNA using PCR, transferring the amplicon target DNA to amembrane in a dot-blot format, hybridizing a labeled oligonucleotideprobe to the amplicon target, and observing the hybridization dots byautoradiography.

Single nucleotide polymorphisms (SNP) are markers that consist of ashared sequence differentiated on the basis of a single nucleotide.Typically, this distinction is detected by differential migrationpatterns of an amplicon comprising the SNP on, e.g., an acrylamide gel.However, alternative modes of detection, such as hybridization, e.g.,ASH, or RFLP analysis are also appropriate.

Isozyme markers can be employed as genetic markers, e.g., to trackmarkers other than the tolerance markers herein, or to track isozymemarkers linked to the markers herein. Isozymes are multiple forms ofenzymes that differ from one another in their amino acid sequence, andtherefore their nucleic acid sequences. Some isozymes are multimericenzymes containing slightly different subunits. Other isozymes areeither multimeric or monomeric but have been cleaved from the proenzymeat different sites in the amino acid sequence. Isozymes can becharacterized and analyzed at the protein level, or alternatively,isozymes, which differ at the nucleic acid level, can be determined. Insuch cases any of the nucleic acid based methods described herein can beused to analyze isozyme markers.

Probe/Primer Synthesis Methods

In general, synthetic methods for making oligonucleotides, includingprobes, primers, molecular beacons, PNAs, LNAs (locked nucleic acids),etc., are well known. For example, oligonucleotides can be synthesizedchemically according to the solid phase phosphoramidite triester methoddescribed by Beaucage and Caruthers (1981) Tetrahedron Letts22:1859-1862, e.g., using a commercially available automatedsynthesizer, e.g., as described in Needham-VanDevanter et al. (1984)Nucleic Acids Res. 12:6159-6168. Oligonucleotides, including modifiedoligonucleotides can also be ordered from a variety of commercialsources known to persons of skill. There are many commercial providersof oligo synthesis services, and thus this is a broadly accessibletechnology. Any nucleic acid can be custom ordered from any of a varietyof commercial sources, such as The Midland Certified Reagent Company(mcrc@oligos.com), The Great American Gene Company (genco.com),ExpressGen Inc. (expressgen.com), Operon Technologies Inc. (Alameda,Calif.) and many others. Similarly, PNAs can be custom ordered from anyof a variety of sources, such as PeptidoGenic (pkim@ccnet.com), HTIBio-products, inc. (htibio.com), BMA Biomedicals Ltd (U.K.), Bio.Synthesis, Inc., and many others.

In Silico Marker Detection

In alternative embodiments, in silico methods can be used to detect themarker loci of interest. For example, the sequence of a nucleic acidcomprising the marker locus of interest can be stored in a computer. Thedesired marker locus sequence or its homolog can be identified using anappropriate nucleic acid search algorithm as provided by, for example,in such readily available programs as BLAST, or even simple wordprocessors.

Amplification Primers for Marker Detection

In some examples, molecular markers are detected using a suitablePCR-based detection method, where the size or sequence of the PCRamplicon is indicative of the absence or presence of the marker (e.g., aparticular marker allele). In these types of methods, PCR primers arehybridized to the conserved regions flanking the polymorphic markerregion. Suitable primers can be designed using any suitable method. Itis not intended that the invention be limited to any particular primeror primer pair. For example, primers can be designed using any suitablesoftware program, such as LASERGENE®.

In some examples, the primers are radiolabelled, or labeled by anysuitable means (e.g., using a non-radioactive fluorescent tag), to allowfor rapid visualization of the different size amplicons following anamplification reaction without any additional labeling step orvisualization step. In some examples, the primers are not labeled, andthe amplicons are visualized following their size resolution, e.g.,following agarose gel electrophoresis. In some examples, ethidiumbromide staining of the PCR amplicons following size resolution allowsvisualization of the different size amplicons.

The primers used to amplify the marker loci and alleles herein are notlimited to amplifying the entire region of the relevant locus. In someexamples, marker amplification produces an amplicon at least 20nucleotides in length, or alternatively, at least 50 nucleotides inlength, or alternatively, at least 100 nucleotides in length, oralternatively, at least 200 nucleotides in length, or up to andincluding the full length of the amplicon.

Marker Assisted Selection and Breeding of Plants

A primary motivation for development of molecular markers in cropspecies is the potential for increased efficiency in plant breedingthrough marker assisted selection (MAS). Genetic markers are used toidentify plants that contain a desired genotype at one or more loci, andthat are expected to transfer the desired genotype, along with a desiredphenotype to their progeny. Genetic markers can be used to identifyplants that contain a desired genotype at one locus, or at severalunlinked or linked loci (e.g., a haplotype), and that would be expectedto transfer the desired genotype, along with a desired phenotype totheir progeny. Means to identify plants, particularly soybean plants,that are tolerant, or that exhibit improved tolerance toprotoporphyrinogen oxidase inhibitors are provided, for example byidentifying plants having a specified marker loci e.g., for linkagegroup L: SATT495, P10649C-3, SATT182, SATT388, SATT313, SATT613 (andother markers above SATT613), S08102-1-Q1, S08103-1-Q1. S08104-1-Q1,S08106-1-Q1, S08107-1-Q1, S08107-1-Q1, S08109-1-Q1, S08110-1-Q1,S08111-1-Q1, S08115-2-Q1, S08117-1-Q1, S08119-1-Q1, S08116-1-Q1,S08112-1-Q1, S08108-1-Q1, S08101-4-Q1, S08101-1-Q1, S08101-2-Q1, andS08101-3-Q1, and/or for linkage group N: Sat_(—)379, SCT_(—)195,SATT631, S60167-TB, SATT675, SATT624, SATT080, and SATT387 (and othermarkers above SATT387). Similarly, by identifying plants lacking thedesired marker locus, non-tolerant or less tolerant plants can beidentified, and, e.g., eliminated from subsequent crosses. Similarly,these marker loci can be introgressed into any desired genomicbackground, germplasm, plant, line, variety, etc., as part of an overallMAS breeding program designed to enhance soybean yield.

In general, the application of MAS uses the identification of apopulation of tolerant plants and genetic mapping of the tolerancetrait. Polymorphic loci in the vicinity of the mapped tolerance traitare chosen as potential tolerance markers. Typically, a marker locusclosest to the tolerance locus is a preferred marker. Linkage analysisis then used to determine which polymorphic marker allele sequencedemonstrates a statistical likelihood of co-segregation with thetolerant phenotype (thus, a “tolerance marker allele”). Followingidentification of a marker allele for co-segregation with the toleranceallele, it is possible to use this marker for rapid, accurate screeningof plant lines for the tolerance allele without the need to grow theplants through their life cycle and await phenotypic evaluations, andfurthermore, permits genetic selection for the particular toleranceallele even when the molecular identity of the actual tolerance QTL isanonymous. Tissue samples can be taken, for example, from the first leafof the plant and screened with the appropriate molecular marker, andwithin days it is determined which progeny will advance. Linked markersalso remove the impact of environmental factors that can often influencephenotypic expression.

After a desired phenotype (e.g., tolerance to protoporphyrinogen oxidaseinhibitors) and a polymorphic chromosomal marker locus are determined tocosegregate, the polymorphic marker locus can be used to select formarker alleles that segregate with the desired tolerance phenotype. Thisgeneral process is typically called marker-assisted selection (MAS). Inbrief, a nucleic acid corresponding to the marker nucleic acid isdetected in a biological sample from a plant to be selected. Thisdetection can take the form of hybridization of a probe nucleic acid toa marker allele or amplicon thereof, e.g., using allele-specifichybridization, Southern analysis, northern analysis, in situhybridization, hybridization of primers followed by PCR amplification ofa region of the marker, or the like. After the presence (or absence) ofa particular marker in the biological sample is verified, the plant isselected, e.g., used to make progeny plants by selective breeding.

Soybean plant breeders desire combinations of tolerance loci with genesfor high yield and other desirable traits to develop improved soybeanvarieties. Screening large numbers of samples by non-molecular methods(e.g., trait evaluation in soybean plants) can be expensive, timeconsuming, and unreliable. Use of the polymorphic markers describedherein genetically linked to tolerance loci provide effective methodsfor selecting tolerant varieties in breeding programs. For example, oneadvantage of marker-assisted selection over field evaluations fortolerance is that MAS can be done at any time of year, regardless of thegrowing season. Moreover, environmental effects are largely irrelevantto marker-assisted selection.

When a population is segregating for multiple loci affecting one ormultiple traits, e.g., multiple loci involved in tolerance, or multipleloci each involved in tolerance or tolerance to different herbicides,the efficiency of MAS compared to phenotypic screening becomes evengreater, because all of the loci can be evaluated in the lab togetherfrom a single sample of DNA. In the present instance, for linkage groupL: SATT495, P10649C-3, SATT182, SATT388, SATT313, SATT613 (or othermarkers above SATT613), S08102-1-Q1, S08103-1-Q1. S08104-1-Q1,S08106-1-Q1, S08107-1-Q1, S08107-1-Q1, S08109-1-Q1, S08110-1-Q1,S08111-1-Q1, S08115-2-Q1, S08117-1-Q1, S08119-1-Q1, 508116-1-Q1,S08112-1-Q1, S08108-1-Q1, S08101-4-Q1, S08101-1-Q1, S08101-2-Q1, andS08101-3-Q1; and for linkage group N: Sat_(—)379, SCT_(—)195, SATT631,S60167-TB, SATT675, SATT624, SATT080, and SATT387 (or other markersabove SATT387) markers, and markers for other traits, transgenes, and/orloci can be assayed simultaneously or sequentially in a single sample orpopulation of samples.

Another use of MAS in plant breeding is to assist the recovery of therecurrent parent genotype by backcross breeding. Backcross breeding isthe process of crossing a progeny back to one of its parents or parentlines. Backcrossing is usually done for the purpose of introgressing oneor a few loci from a donor parent (e.g., a parent comprising desirabletolerance marker loci) into an otherwise desirable genetic backgroundfrom the recurrent parent (e.g., an otherwise high yielding soybeanline). The more cycles of backcrossing that are done, the greater thegenetic contribution of the recurrent parent to the resultingintrogressed variety. This is often necessary, because tolerant plantsmay be otherwise undesirable, e.g., due to low yield, low fecundity, orthe like. In contrast, strains which are the result of intensivebreeding programs may have excellent yield, fecundity or the like,merely being deficient in one desired trait such as tolerance toprotoporphyrinogen oxidase inhibitors.

The presence and/or absence of a particular genetic marker or allele,e.g., for linkage group L: SATT495, P10649C-3, SATT182, SATT388,SATT313, SATT613 (including markers above SATT613), S08102-1-Q1,S08103-1-Q1, S08104-1-Q1, S08106-1-Q1, S08107-1-Q1, S08107-1-Q1,S08109-1-Q1, S08110-1-Q1, S08111-1-Q1, S08115-2-Q1, S08117-1-Q1,S08119-1-Q1, S08116-1-Q1, S08112-1-Q1, S08108-1-Q1, S08101-4-Q1,S08101-1-Q1, S08101-2-Q1, and S08101-3-Q1, and for linkage group N:Sat_(—)379, SCT_(—)195, SATT631, S60167-TB, SATT675, SATT624, SATT080,and SATT387 (including markers above SATT387) in the genome of a plantexhibiting a preferred phenotypic trait is made by any method notedherein. If the nucleic acids from the plant are positive for a desiredgenetic marker, the plant can be self fertilized to create a truebreeding line with the same genotype, or it can be crossed with a plantwith the same marker or with other desired characteristics to create asexually crossed hybrid generation.

Introgression of Favorable Alleles—Efficient Crossing of ToleranceMarkers into Other Lines

One application of MAS is to use the tolerance or improved tolerancemarkers to increase the efficiency of an introgression or backcrossingeffort aimed at introducing a tolerance QTL into a desired (typicallyhigh yielding) background. In marker assisted backcrossing of specificmarkers (and associated QTL) from a donor source, e.g., to an elitegenetic background, one selects among progeny or backcross progeny forthe donor trait.

Thus, the markers and methods can be utilized to guide marker assistedselection or breeding of soybean varieties with the desired complement(set) of allelic forms of chromosome segments associated with herbicidetolerance as well as markers associated with superior agronomicperformance (tolerance, along with any other available markers foryield, disease tolerance, etc.). Any of the disclosed marker alleles canbe introduced into a soybean line via introgression, by traditionalbreeding (or introduced via transformation, or both) to yield a soybeanplant with superior agronomic performance. The number of allelesassociated with tolerance that can be introduced or be present in asoybean plant ranges from 1 to the number of alleles disclosed herein,each integer of which is incorporated herein as if explicitly recited.

Methods of making a progeny soybean plant and these progeny soybeanplants having tolerance to PPO inhibitors are provided. These methodscomprise crossing a first parent soybean plant with a second soybeanplant and growing the female soybean plant under plant growth conditionsto yield soybean plant progeny. Such soybean plant progeny can beassayed for alleles associated with tolerance and, thereby, the desiredprogeny selected. Such progeny plants or seed can be sold commerciallyfor soybean production, used for food, processed to obtain a desiredconstituent of the soybean, or further utilized in subsequent rounds ofbreeding. At least one of the first or second soybean plants is asoybean plant comprising at least one of the allelic forms of themarkers provided, such that the progeny are capable of inheriting theallele.

Inheritance of the desired tolerance allele can be traced, such as fromprogenitor or descendant lines in the subject soybean plants pedigreesuch that the number of generations separating the soybean plants beingsubject to the methods will generally be from 1 to 20, commonly 1 to 5,and typically 1, 2, or 3 generations of separation, and quite often adirect descendant or parent of the soybean plant will be subject to themethod (i.e., 1 generation of separation).

Methods for Identifying Protoporphyrinogen Oxidase Inhibitor TolerantSoybean Plants

Experienced plant breeders can recognize tolerant soybean plants in thefield, and can select the tolerant individuals or populations forbreeding purposes or for propagation. In this context, the plant breederrecognizes tolerant, and non-tolerant soybean plants.

The screening and selection may also be performed by exposing plantscontaining said progeny germplasm to protoporphyrinogen oxidaseinhibitors in an assay and selecting those plants showing tolerance toprotoporphyrinogen oxidase inhibitors as containing soybean germplasminto which germplasm having tolerance to protoporphyrinogen oxidaseinhibitors derived from the QTL mapped to linkage group L and/or N hasbeen introgressed. The live assay may be any such assay known to theart, e.g., Taylor-Lovell et al. (2001) Weed Tech 15:95-102.

However, plant tolerance is a phenotypic spectrum consisting of extremesof high tolerance to non-tolerance with a continuum of intermediatetolerance phenotypes. Evaluation of these intermediate phenotypes usingreproducible assays are of value to scientists who seek to identifygenetic loci that impart tolerance, conduct marker assisted selectionfor tolerant population, and for introgression techniques to breed atolerance trait into an elite soybean line, for example. Describing thecontinuum of tolerance can be done using any known scoring system orderivative thereof, including the scoring systems described in Examples1-4.

Automated Detection/Correlation Systems

In some examples, the methods include an automated system for detectingmarkers and or correlating the markers with a desired phenotype (e.g.,tolerance). Thus, a typical system can include a set of marker probes orprimers configured to detect at least one favorable allele of one ormore marker locus associated with tolerance or improved tolerance toprotoporphyrinogen oxidase inhibitors. These probes or primers areconfigured to detect the marker alleles noted in the tables and examplesherein, e.g., using any available allele detection format, e.g., solidor liquid phase array based detection, microfluidic-based sampledetection, etc.

In some examples markers involving linkage group L are used. In someexamples a marker closely linked to the marker locus of SATT495,P10649C-3, SATT182, SATT388, SATT313, SATT613, S08102-1-Q1,S08103-1-Q1S08104-1-Q1, S08106-1-Q1, S08107-1-Q1, S08107-1-Q1,S08109-1-Q1, S08110-1-Q1, S08111-1-Q1, S08115-2-Q1, S08117-1-Q1,S08119-1-Q1, S08116-1-Q1, S08112-1-Q1, S08108-1-Q1, S08101-4-Q1,S08101-1-Q1, S08101-2-Q1, and S08101-3-Q1 is used, and the probe set isconfigured to detect the closely linked marker(s). In some examples, themarker locus is SATT495, P10649C-3, SATT182, SATT388, SATT313, SATT613(or another marker above SATT613), S08102-1-Q1, S08103-1-Q1.S08104-1-Q1, S08106-1-Q1, S08107-1-Q1, S08107-1-Q1, S08109-1-Q1,S08110-1-Q1, S08111-1-Q1, S08115-2-Q1, S08117-1-Q1, S08119-1-Q1,S08116-1-Q1, S08112-1-Q1, S08108-1-Q1, S08101-4-Q1, S08101-1-Q1,S08101-2-Q1, and S08101-3-Q1 and the probe set is configured to detectthe locus. Similarly, alleles of SATT495, P10649C-3, SATT182, SATT388,SATT313, and SATT613 can be detected.

In some examples markers involving linkage group N are used. In someexamples a marker closely linked to the marker locus of Sat_(—)379,SCT_(—)195, SATT631, S60167-TB, SATT675, SATT624, SATT080, and SATT387(or another marker above SATT387) is used, and the probe set isconfigured to detect the closely linked marker(s). In some examples themarker locus is Sat_(—)379, SCT_(—)195, SATT631, S60167-TB, SATT675,SATT624, SATT080, and SATT387 and the probe set is configured to detectthe locus. Similarly, alleles of Sat_(—)379, SCT_(—)195, SATT631,S60167-TB, SATT675, SATT624, SATT080, and SATT387 can be detected.

The typical system includes a detector that is configured to detect oneor more signal outputs from the set of marker probes or primers, oramplicon thereof, thereby identifying the presence or absence of theallele. A wide variety of signal detection apparatus are available,including photo multiplier tubes, spectrophotometers, CCD arrays, arraysand array scanners, scanning detectors, phototubes and photodiodes,microscope stations, galvo-scans, microfluidic nucleic acidamplification detection appliances and the like. The preciseconfiguration of the detector will depend, in part, on the type of labelused to detect the marker allele, as well as the instrumentation that ismost conveniently obtained for the user. Detectors that detectfluorescence, phosphorescence, radioactivity, pH, charge, absorbance,luminescence, temperature, magnetism or the like can be used. Typicaldetector examples include light (e.g., fluorescence) detectors orradioactivity detectors. For example, detection of a light emission(e.g., a fluorescence emission) or other probe label is indicative ofthe presence or absence of a marker allele. Fluorescent detection isgenerally used for detection of amplified nucleic acids (however,upstream and/or downstream operations can also be performed onamplicons, which can involve other detection methods). In general, thedetector detects one or more label (e.g., light) emission from a probelabel, which is indicative of the presence or absence of a markerallele. The detector(s) optionally monitors one or a plurality ofsignals from an amplification reaction. For example, the detector canmonitor optical signals which correspond to “real time” amplificationassay results.

System instructions that correlate the presence or absence of thefavorable allele with the predicted tolerance are also provided. Forexample, the instructions can include at least one look-up table thatincludes a correlation between the presence or absence of the favorablealleles and the predicted tolerance or improved tolerance. The preciseform of the instructions can vary depending on the components of thesystem, e.g., they can be present as system software in one or moreintegrated unit of the system (e.g., a microprocessor, computer orcomputer readable medium), or can be present in one or more units (e.g.,computers or computer readable media) operably coupled to the detector.As noted, in one typical example, the system instructions include atleast one look-up table that includes a correlation between the presenceor absence of the favorable alleles and predicted tolerance or improvedtolerance. The instructions also typically include instructionsproviding a user interface with the system, e.g., to permit a user toview results of a sample analysis and to input parameters into thesystem.

The system typically includes components for storing or transmittingcomputer readable data representing or designating the alleles detectedby the methods, e.g., in an automated system. The computer readablemedia can include cache, main, and storage memory and/or otherelectronic data storage components (hard drives, floppy drives, storagedrives, etc.) for storage of computer code. Data representing allelesdetected by the methods can also be electronically, optically,magnetically o transmitted in a computer data signal embodied in atransmission medium over a network such as an intranet or internet orcombinations thereof. The system can also or alternatively transmit datavia wireless, IR, or other available transmission alternatives.

During operation, the system typically comprises a sample that is to beanalyzed, such as a plant tissue, or material isolated from the tissuesuch as genomic DNA, amplified genomic DNA, cDNA, amplified cDNA, RNA,amplified RNA, or the like.

The phrase “allele detection/correlation system” refers to a system inwhich data entering a computer corresponds to physical objects orprocesses external to the computer, e.g., a marker allele, and a processthat, within a computer, causes a physical transformation of the inputsignals to different output signals. In other words, the input data,e.g., amplification of a particular marker allele is transformed tooutput data, e.g., the identification of the allelic form of achromosome segment. The process within the computer is a set ofinstructions, or “program,” by which positive amplification orhybridization signals are recognized by the integrated system andattributed to individual samples as a genotype. Additional programscorrelate the identity of individual samples with phenotypic values ormarker alleles, e.g., statistical methods. In addition there arenumerous e.g., C/C++ programs for computing, Delphi and/or Java programsfor GUI interfaces, and productivity tools (e.g., Microsoft Excel and/orSigmaPlot) for charting or creating look up tables of relevantallele-trait correlations. Other useful software tools in the context ofthe integrated systems include statistical packages such as SAS,Genstat, Matlab, Mathematica, and S-Plus and genetic modeling packagessuch as QU-GENE. Furthermore, additional programming languages such asvisual basic are also suitably employed in the integrated systems.

For example, tolerance marker allele values assigned to a population ofprogeny descending from crosses between elite lines are recorded in acomputer readable medium, thereby establishing a database correspondingtolerance alleles with unique identifiers for members of the populationof progeny. Any file or folder, whether custom-made or commerciallyavailable (e.g., from Oracle or Sybase) suitable for recording data in acomputer readable medium is acceptable as a database. Data regardinggenotype for one or more molecular markers, e.g., ASH, SSR, RFLP, RAPD,AFLP, SNP, isozyme markers or other markers as described herein, aresimilarly recorded in a computer accessible database. Optionally, markerdata is obtained using an integrated system that automates one or moreaspects of the assay (or assays) used to determine marker(s) genotype.In such a system, input data corresponding to genotypes for molecularmarkers are relayed from a detector, e.g., an array, a scanner, a CCD,or other detection device directly to files in a computer readablemedium accessible to the central processing unit. A set of systeminstructions (typically embodied in one or more programs) encoding thecorrelations between tolerance and the alleles of the invention is thenexecuted by the computational device to identify correlations betweenmarker alleles and predicted trait phenotypes.

Typically, the system also includes a user input device, such as akeyboard, a mouse, a touchscreen, or the like, for, e.g., selectingfiles, retrieving data, reviewing tables of maker information, etc., andan output device (e.g., a monitor, a printer, etc.) for viewing orrecovering the product of the statistical analysis.

Integrated systems comprising a computer or computer readable mediumcomprising set of files and/or a database with at least one data setthat corresponds to the marker alleles herein are provided. The systemsoptionally also includes a user interface allowing a user to selectivelyview one or more of these databases. In addition, standard textmanipulation software such as word processing software (e.g., MicrosoftWord™ or Corel Wordperfect™) and database or spreadsheet software (e.g.,spreadsheet software such as Microsoft Excel™, Corel Quattro Pro™, ordatabase programs such as Microsoft Access™ or Paradox™) can be used inconjunction with a user interface (e.g., a GUI in a standard operatingsystem such as a Windows, Macintosh, Unix or Linux system) to manipulatestrings of characters corresponding to the alleles or other features ofthe database.

The systems optionally include components for sample manipulation, e.g.,incorporating robotic devices. For example, a robotic liquid controlarmature for transferring solutions (e.g., plant cell extracts) from asource to a destination, e.g., from a microtiter plate to an arraysubstrate, is optionally operably linked to the digital computer (or toan additional computer in the integrated system). An input device forentering data to the digital computer to control high throughput liquidtransfer by the robotic liquid control armature and, optionally, tocontrol transfer by the armature to the solid support is commonly afeature of the integrated system. Many such automated robotic fluidhandling systems are commercially available. For example, a variety ofautomated systems are available from Caliper Technologies (Hopkinton,Mass.), which utilize various Zymate systems, which typically include,e.g., robotics and fluid handling modules. Similarly, the common ORCA®robot, which is used in a variety of laboratory systems, e.g., formicrotiter tray manipulation, is also commercially available, e.g., fromBeckman Coulter, Inc. (Fullerton, Calif.). As an alternative toconventional robotics, microfluidic systems for performing fluidhandling and detection are now widely available, e.g., from CaliperTechnologies Corp. (Hopkinton, Mass.) and Agilent technologies (PaloAlto, Calif.).

Systems for molecular marker analysis can include a digital computerwith one or more of high-throughput liquid control software, imageanalysis software for analyzing data from marker labels, datainterpretation software, a robotic liquid control armature fortransferring solutions from a source to a destination operably linked tothe digital computer, an input device (e.g., a computer keyboard) forentering data to the digital computer to control high throughput liquidtransfer by the robotic liquid control armature and, optionally, animage scanner for digitizing label signals from labeled probeshybridized, e.g., to markers on a solid support operably linked to thedigital computer. The image scanner interfaces with the image analysissoftware to provide a measurement of, e.g., nucleic acid probe labelintensity upon hybridization to an arrayed sample nucleic acidpopulation (e.g., comprising one or more markers), where the probe labelintensity measurement is interpreted by the data interpretation softwareto show whether, and to what degree, the labeled probe hybridizes to amarker nucleic acid (e.g., an amplified marker allele). The data soderived is then correlated with sample identity, to determine theidentity of a plant with a particular genotype(s) for particular markersor alleles, e.g., to facilitate marker assisted selection of soybeanplants with favorable allelic forms of chromosome segments involved inagronomic performance (e.g., tolerance or improved tolerance).

Optical images, e.g., hybridization patterns viewed (and, optionally,recorded) by a camera or other recording device (e.g., a photodiode anddata storage device) are optionally further processed in any of theembodiments herein, e.g., by digitizing the image and/or storing andanalyzing the image on a computer. A variety of commercially availableperipheral equipment and software is available for digitizing, storingand analyzing a digitized video or digitized optical image.

Positional Cloning

The molecular marker loci and alleles associated with tolerance to PPOinhibitors, e.g., SATT495, P10649C-3, SATT182, S03859-1, S00224-1,SATT388, SATT313, and SATT613 (including markers above SATT613),S08102-1-Q1, S08103-1-Q1. S08104-1-Q1, S08106-1-Q1, S08107-1-Q1,S08107-1-Q1, S08109-1-Q1, S08110-1-Q1, S08111-1-Q1, S08115-2-Q1,S08117-1-Q1, S08119-1-Q1, S08116-1-Q1, S08112-1-Q1, S08108-1-Q1,S08101-4-Q1, S08101-1-Q1, S08101-2-Q1, or S08101-3-Q1 can be used, asindicated previously, to identify a tolerance QTL, which can be clonedby well-established procedures, e.g., as described in detail in Ausubel,Berger and Sambrook, herein.

These tolerance clones are first identified by their genetic linkage tomarkers provided herein. Isolation of a nucleic acid of interest isachieved by any number of methods as discussed in detail in suchreferences as Ausubel, Berger and Sambrook, herein, and Clark, ed.(1997) Plant Molecular Biology: A Laboratory Manual Springer-Verlag,Berlin.

For example, “positional gene cloning” uses the proximity of a tolerancemarker to physically define an isolated chromosomal fragment containinga tolerance QTL gene. The isolated chromosomal fragment can be producedby such well known methods as digesting chromosomal DNA with one or morerestriction enzymes, or by amplifying a chromosomal region in apolymerase chain reaction (PCR), or any suitable alternativeamplification reaction. The digested or amplified fragment is typicallyligated into a vector suitable for replication, and, e.g., expression,of the inserted fragment. Markers that are adjacent to an open readingframe (ORF) associated with a phenotypic trait can hybridize to a DNAclone (e.g., a clone from a genomic DNA library), thereby identifying aclone on which an ORF (or a fragment of an ORF) is located. If themarker is more distant, a fragment containing the open reading frame isidentified by successive rounds of screening and isolation of cloneswhich together comprise a contiguous sequence of DNA, a process termed“chromosome walking”, resulting in a “contig” or “contig map.” Protocolssufficient to guide one of skill through the isolation of clonesassociated with linked markers are found in, e.g. Berger, Sambrook andAusubel, all herein.

Variant sequences have a high degree of sequence similarity. Forpolynucleotides, conservative variants include those sequences that,because of the degeneracy of the genetic code, encode the amino acidsequence of one of the native recombinase polypeptides. Variants such asthese can be identified with the use of well-known molecular biologytechniques, as, for example, with polymerase chain reaction (PCR) andhybridization techniques. Variant polynucleotides also includesynthetically derived nucleotide sequences, such as those generated, forexample, by using site-directed mutagenesis but which still encode arecombinase protein. Generally, variants of a particular polynucleotidewill have at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%,85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequenceidentity to that particular polynucleotide as determined by knownsequence alignment programs and parameters.

Variants of a particular polynucleotide (the reference nucleotidesequence) can also be evaluated by comparison of the percent sequenceidentity between the polypeptide encoded by a variant polynucleotide andthe polypeptide encoded by the reference polynucleotide. Thus, forexample, isolated polynucleotides that encode a polypeptide with a givenpercent sequence identity to the recombinase are known. Percent sequenceidentity between any two polypeptides can be calculated using sequencealignment programs and parameters described. Where any given pair ofpolynucleotides is evaluated by comparison of the percent sequenceidentity shared by the two polypeptides they encode, the percentsequence identity between the two encoded polypeptides is at least about40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99% or more sequence identity.

A variant protein is intended a protein derived from the native proteinby deletion, addition, and/or substitution of one or more amino acids tothe N-terminal, internal region(s), and/or C-terminal end of the nativeprotein. Variant proteins are biologically active, that is they continueto possess the desired biological activity of the native protein, forexample a variant recombinase will implement a recombination eventbetween appropriate recombination sites. Such variants may result from,for example, genetic polymorphism or from human manipulation.Biologically active variants of a native recombinase protein will haveat least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity tothe amino acid sequence for the native protein as determined by knownsequence alignment programs and parameters. A biologically activevariant of a protein may differ from that protein by as few as 1-15amino acid residues, as few as 1-10, such as 6-10, as few as 5, as fewas 4, 3, 2, or even 1 amino acid residue.

Sequence relationships can be analyzed and described usingcomputer-implemented algorithms. The sequence relationship between twoor more polynucleotides, or two or more polypeptides can be determinedby generating the best alignment of the sequences, and scoring thematches and the gaps in the alignment, which yields the percent sequenceidentity, and the percent sequence similarity. Polynucleotiderelationships can also be described based on a comparison of thepolypeptides each encodes. Many programs and algorithms for thecomparison and analysis of sequences are available.

Unless otherwise stated, sequence identity/similarity values providedherein refer to the value obtained using GAP Version 10 (GCG, Accelrys,San Diego, Calif.) using the following parameters: % identity and %similarity for a nucleotide sequence using a gap creation penalty weightof 50 and a gap length extension penalty weight of 3, and thenwsgapdna.cmp scoring matrix; % identity and % similarity for an aminoacid sequence using a GAP creation penalty weight of 8 and a gap lengthextension penalty of 2, and the BLOSUM62 scoring matrix (Henikoff &Henikoff (1989) Proc Natl Acad Sci USA 89:10915).

GAP uses the algorithm of Needleman & Wunsch (1970) J Mol Biol48:443-453, to find an alignment of two complete sequences thatmaximizes the number of matches and minimizes the number of gaps. GAPconsiders all possible alignments and gap positions and creates thealignment with the largest number of matched bases and the fewest gaps.It allows for the provision of a gap creation penalty and a gapextension penalty in units of matched bases. GAP must make a profit ofgap creation penalty number of matches for each gap it inserts. If a gapextension penalty greater than zero is chosen, GAP must, in addition,make a profit for each gap inserted of the length of the gap times thegap extension penalty. GAP presents one member of the family of bestalignments.

Sequence identity, or identity, is a measure of the residues in the twosequences that are the same when aligned for maximum correspondence.Sequences, particularly polypeptides, that differ by conservativesubstitutions are said to have sequence similarity or similarity. Meansfor making this adjustment are known, and typically involve scoring aconservative substitution as a partial rather than a full mismatch. Forexample, where an identical amino acid is given a score of 1 and anon-conservative substitution is given a score of zero, a conservativesubstitution is given a score between zero and 1. The scoring ofconservative substitutions is calculated using the selected scoringmatrix (BLOSUM62 by default for GAP).

Proteins may be altered in various ways including amino acidsubstitutions, deletions, truncations, and insertions. Methods for suchmanipulations are generally known. For example, amino acid sequencevariants of the recombinase proteins can be prepared by mutations in theDNA. Methods for mutagenesis and nucleotide sequence alterations includefor example, Kunkel (1985) Proc Natl Acad Sci USA 82:488-492; Kunkel etal. (1987) Methods in Enzymol 154:367-382; U.S. Pat. No. 4,873,192;Walker & Gaastra, eds. (1983) Techniques in Molecular Biology (MacMillanPublishing Company, New York) and the references cited therein. Guidanceas to appropriate amino acid substitutions that do not affect biologicalactivity of the protein of interest may be found in the model of Dayhoffet al. (1978) Atlas of Protein Sequence and Structure (Natl Biomed ResFound, Washington, D.C.). Conservative substitutions, such as exchangingone amino acid with another having similar properties, may bepreferable.

Generation of Transgenic Cells and Plants

The present invention also relates to host cells and organisms which aretransformed with nucleic acids corresponding to tolerance QTL identifiedherein. For example, such nucleic acids include chromosome intervals(e.g., genomic fragments), ORFs and/or cDNAs that encode a tolerance orimproved tolerance trait. Additionally, production of polypeptides thatprovide tolerance or improved tolerance by recombinant techniques areprovided.

General texts which describe molecular biological techniques for thecloning and manipulation of nucleic acids and production of encodedpolypeptides include Berger and Kimmel, Guide to Molecular CloningTechniques, Methods in Enzymology volume 152 Academic Press, Inc., SanDiego, Calif. (Berger); Sambrook et al., Molecular Cloning—A LaboratoryManual (3rd Ed.), Vol. 1-3, Cold Spring Harbor Laboratory, Cold SpringHarbor, N.Y., 2001 (“Sambrook”) and Current Protocols in MolecularBiology, F. M. Ausubel et al., eds., Current Protocols, a joint venturebetween Greene Publishing Associates, Inc. and John Wiley & Sons, Inc.,(supplemented through 2004 or later) (“Ausubel”)). These texts describemutagenesis, the use of vectors, promoters and many other relevanttopics related to, e.g., the generation of clones that comprise nucleicacids of interest, e.g., marker loci, marker probes, QTL that segregatewith marker loci, etc.

Host cells are genetically engineered (e.g., transduced, transfected,transformed, etc.) with the vectors (e.g., vectors, such as expressionvectors which comprise an ORF derived from or related to a toleranceQTL) which can be, for example, a cloning vector, a shuttle vector or anexpression vector. Such vectors are, for example, in the form of aplasmid, a phagemid, an agrobacterium, a virus, a naked polynucleotide(linear or circular), or a conjugated polynucleotide. Vectors can beintroduced into bacteria, especially for the purpose of propagation andexpansion. The vectors are also introduced into plant tissues, culturedplant cells or plant protoplasts by a variety of standard methods knownin the art, including but not limited to electroporation (From et al.(1985) Proc. Natl. Acad. Sci. USA 82; 5824), infection by viral vectorssuch as cauliflower mosaic virus (CaMV) (Hohn et al. (1982) MolecularBiology of Plant Tumors (Academic Press, New York, pp. 549-560; HowellU.S. Pat. No. 4,407,956), high velocity ballistic penetration by smallparticles with the nucleic acid either within the matrix of small beadsor particles, or on the surface (Klein et al. (1987) Nature 327; 70),use of pollen as vector (WO 85/01856), or use of Agrobacteriumtumefaciens or A. rhizogenes carrying a T-DNA plasmid in which DNAfragments are cloned. The T-DNA plasmid is transmitted to plant cellsupon infection by Agrobacterium tumefaciens, and a portion is stablyintegrated into the plant genome (Horsch et al. (1984) Science 233; 496;Fraley et al. (1983) Proc. Natl. Acad. Sci. USA 80; 4803). Additionaldetails regarding nucleic acid introduction methods are found inSambrook, Berger and Ausubel, infra. The method of introducing a nucleicacid into a host cell is not critical, and therefore should not belimited to any particular method for introducing exogenous geneticmaterial into a host cell. Thus, any suitable method, e.g., includingbut not limited to the methods provided herein, which provides foreffective introduction of a nucleic acid into a cell or protoplast canbe employed.

The engineered host cells can be cultured in conventional nutrient mediamodified as appropriate for such activities as, for example, activatingpromoters or selecting transformants. These cells can optionally becultured into transgenic plants. In addition to Sambrook, Berger andAusubel, all infra. Plant regeneration from cultured protoplasts isdescribed in Evans et al. (1983) “Protoplast Isolation and Culture,”Handbook of Plant Cell Cultures 1, 124-176 (MacMillan Publishing Co.,New York; Davey (1983) “Recent Developments in the Culture andRegeneration of Plant Protoplasts,” Protoplasts, pp. 12-29, (Birkhauser,Basel); Dale (1983) “Protoplast Culture and Plant Regeneration ofCereals and Other Recalcitrant Crops,” Protoplasts pp. 31-41,(Birkhauser, Basel); Binding (1985) “Regeneration of Plants,” PlantProtoplasts, pp. 21-73, (CRC Press, Boca Raton, Fla.). Additionaldetails regarding plant cell culture and regeneration include Payne etal. (1992) Plant Cell and Tissue Culture in Liquid Systems John Wiley &Sons, Inc. New York, N.Y.; Gamborg and Phillips (eds) (1995) Plant Cell,Tissue and Organ Culture; Fundamental Methods Springer Lab Manual,Springer-Verlag (Berlin Heidelberg New York) and Plant Molecular Biology(1993) R. R. D. Croy, Ed. Bios Scientific Publishers, Oxford, U.K. ISBN0 12 198370 6. Cell culture media in general are also set forth in Atlasand Parks (eds) The Handbook of Microbiological Media (1993) CRC Press,Boca Raton, Fla. Additional information for cell culture is found inavailable commercial literature such as the Life Science Research CellCulture Catalogue (1998) from Sigma-Aldrich, Inc (St Louis, Mo.)(“Sigma-LSRCCC”) and, e.g., the Plant Culture Catalogue and supplement(e.g., 1997 or later) also from Sigma-Aldrich, Inc (St Louis, Mo.)(“Sigma-PCCS”).

The production of transgenic organisms is provided, which may bebacteria, yeast, fungi, animals or plants, transduced with the nucleicacids (e.g., nucleic acids comprising the marker loci and/or QTL notedherein). A thorough discussion of techniques relevant to bacteria,unicellular eukaryotes and cell culture is found in referencesenumerated herein and are briefly outlined as follows. Severalwell-known methods of introducing target nucleic acids into bacterialcells are available, any of which may be used. These include: fusion ofthe recipient cells with bacterial protoplasts containing the DNA,treatment of the cells with liposomes containing the DNA,electroporation, microinjection, cell fusions, projectile bombardment(biolistics), carbon fiber delivery, and infection with viral vectors(discussed further, below), etc. Bacterial cells can be used to amplifythe number of plasmids containing DNA constructs. The bacteria are grownto log phase and the plasmids within the bacteria can be isolated by avariety of methods known in the art (see, for instance, Sambrook). Inaddition, a plethora of kits are commercially available for thepurification of plasmids from bacteria. For their proper use, follow themanufacturer's instructions (see, for example, EasyPrep™, FlexiPrep™,both from Pharmacia Biotech; StrataClean™, from Stratagene; and,QIAprep™ from Qiagen). The isolated and purified plasmids are thenfurther manipulated to produce other plasmids, used to transfect plantcells or incorporated into Agrobacterium tumefaciens related vectors toinfect plants. Typical vectors contain transcription and translationterminators, transcription and translation initiation sequences, andpromoters useful for regulation of the expression of the particulartarget nucleic acid. The vectors optionally comprise generic expressioncassettes containing at least one independent terminator sequence,sequences permitting replication of the cassette in eukaryotes, orprokaryotes, or both, (e.g., shuttle vectors) and selection markers forboth prokaryotic and eukaryotic systems. Vectors are suitable forreplication and integration in prokaryotes, eukaryotes, or both. See,Giliman & Smith (1979) Gene 8:81; Roberts et al. (1987) Nature 328:731;Schneider et al. (1995) Protein Expr. Purif. 6435:10; Ausubel, Sambrook,Berger (all infra). A catalogue of bacteria and bacteriophages usefulfor cloning is provided, e.g., by the ATCC, e.g., The ATCC Catalogue ofBacteria and Bacteriophage (1992) Gherna et al. (eds) published by theATCC. Additional basic procedures for sequencing, cloning and otheraspects of molecular biology and underlying theoretical considerationsare also found in Watson et al. (1992) Recombinant DNA, Second Edition,Scientific American Books, N.Y. In addition, essentially any nucleicacid (and virtually any labeled nucleic acid, whether standard ornon-standard) can be custom or standard ordered from any of a variety ofcommercial sources, such as the Midland Certified Reagent Company(Midland, Tex.), The Great American Gene Company (Ramona, Calif.),ExpressGen Inc. (Chicago, Ill.), Operon Technologies Inc. (Alameda,Calif.) and many others.

Introducing Nucleic Acids into Plants

Embodiments include the production of transgenic plants comprising thecloned nucleic acids, e.g., isolated ORFs and cDNAs encoding tolerancegenes. Techniques for transforming plant cells with nucleic acids arewidely available and can be readily adapted. In addition to Berger,Ausubel and Sambrook, all infra, useful general references for plantcell cloning, culture and regeneration include Jones (ed) (1995) PlantGene Transfer and Expression Protocols—Methods in Molecular Biology,Volume 49 Humana Press Towata N.J.; Payne et al. (1992) Plant Cell andTissue Culture in Liquid Systems John Wiley & Sons, Inc. New York, N.Y.(Payne); and Gamborg and Phillips (eds) (1995) Plant Cell, Tissue andOrgan Culture; Fundamental Methods Springer Lab Manual, Springer-Verlag(Berlin Heidelberg N.Y.) (Gamborg). A variety of cell culture media aredescribed in Atlas and Parks (eds) The Handbook of Microbiological Media(1993) CRC Press, Boca Raton, Fla. (Atlas). Additional information forplant cell culture is found in available commercial literature such asthe Life Science Research Cell Culture Catalogue (1998) fromSigma-Aldrich, Inc (St Louis, Mo.) (Sigma-LSRCCC) and, e.g., the PlantCulture Catalogue and supplement (1997) also from Sigma-Aldrich, Inc (StLouis, Mo.) (Sigma-PCCS). Additional details regarding plant cellculture are found in Croy, (ed.) (1993) Plant Molecular Biology, BiosScientific Publishers, Oxford, U.K.

The nucleic acid constructs, e.g., DNA molecules, plasmids, cosmids,artificial chromosomes, DNA and RNA polynucleotides, are introduced intoplant cells, either in culture or in the organs of a plant by a varietyof conventional techniques. Where the sequence is expressed, thesequence is optionally combined with transcriptional and translationalinitiation regulatory sequences which direct the transcription ortranslation of the sequence from the exogenous DNA in the intendedtissues of the transformed plant.

Isolated nucleic acid acids can be introduced into plants according toany of a variety of techniques known in the art. Techniques fortransforming a wide variety of higher plant species are also well knownand described in widely available technical, scientific, and patentliterature. See, e.g., Weising et al. (1988) Ann. Rev. Genet.22:421-477.

The DNA constructs, for example DNA fragments, plasmids, phagemids,cosmids, phage, naked or variously conjugated-DNA polynucleotides,(e.g., polylysine-conjugated DNA, peptide-conjugated DNA,liposome-conjugated DNA, etc.), or artificial chromosomes, can beintroduced directly into the genomic DNA of the plant cell usingtechniques such as electroporation and microinjection of plant cellprotoplasts, or the DNA constructs can be introduced directly to plantcells using ballistic methods, such as DNA particle bombardment.

Microinjection techniques for injecting plant, e.g., cells, embryos,callus and protoplasts, are known in the art and well described in thescientific and patent literature. For example, a number of methods aredescribed in Jones (ed) (1995) Plant Gene Transfer and ExpressionProtocols—Methods in Molecular Biology, Volume 49 Humana Press, Towata,N.J., as well as in the other references noted herein and available inthe literature.

For example, the introduction of DNA constructs using polyethyleneglycol precipitation is described in Paszkowski et al., EMBO J. 3:2717(1984). Electroporation techniques are described in Fromm et al., Proc.Natl. Acad. Sci. USA 82:5824 (1985). Ballistic transformation techniquesare described in Klein et al., Nature 327:70-73 (1987). Additionaldetails are found in Jones (1995) and Gamborg and Phillips (1995),supra, and in U.S. Pat. No. 5,990,387.

Alternatively, Agrobacterium-mediated transformation is employed togenerate transgenic plants. Agrobacterium-mediated transformationtechniques, including disarming and use of binary vectors, are also welldescribed in the scientific literature. See, e.g., Horsch, et al. (1984)Science 233:496; and Fraley et al. (1984) Proc. Natl. Acad. Sci. USA80:4803 and recently reviewed in Hansen and Chilton (1998) CurrentTopics in Microbiology 240:22 and Das (1998) Subcellular Biochemistry29: Plant Microbe Interactions, pp 343-363.

DNA constructs are optionally combined with suitable T-DNA flankingregions and introduced into a conventional Agrobacterium tumefacienshost vector. The virulence functions of the Agrobacterium tumefacienshost will direct the insertion of the construct and adjacent marker intothe plant cell DNA when the cell is infected by the bacteria. See, U.S.Pat. No. 5,591,616. Although Agrobacterium is useful primarily indicots, certain monocots can be transformed by Agrobacterium. Forinstance, Agrobacterium transformation of maize is described in U.S.Pat. No. 5,550,318.

Other methods of transfection or transformation include (1)Agrobacterium rhizogenes-mediated transformation (see, e.g.,Liechtenstein and Fuller (1987) In: Genetic Engineering, vol. 6, P W JRigby, Ed., London, Academic Press; and Liechtenstein; C. P., and Draper(1985) In: DNA Cloning, Vol. II, D. M. Glover, Ed., Oxford, IRI Press;WO 88/02405, published Apr. 7, 1988, describes the use of A. rhizogenesstrain A4 and its Ri plasmid along with A. tumefaciens vectors pARC8 orpARC16 (2) liposome-mediated DNA uptake (see, e.g., Freeman et al.(1984) Plant Cell Physiol. 25:1353), (3) the vortexing method (see,e.g., Kindle (1990) Proc. Natl. Acad. Sci., (USA) 87:1228.

DNA can also be introduced into plants by direct DNA transfer intopollen as described by Zhou et al. (1983) Methods in Enzymology,101:433; D. Hess (1987) Intern Rev. Cytol. 107:367; Luo et al. (1988)Plant Mol. Biol. Reporter 6:165. Expression of polypeptide coding genescan be obtained by injection of the DNA into reproductive organs of aplant as described by Pena et al. (1987) Nature 325:274. DNA can also beinjected directly into the cells of immature embryos and the desiccatedembryos rehydrated as described by Neuhaus et al. (1987) Theor. Appl.Genet. 75:30; and Benbrook et al. (1986) in Proceedings Bio ExpoButterworth, Stoneham, Mass., pp. 27-54. A variety of plant viruses thatcan be employed as vectors are known in the art and include cauliflowermosaic virus (CaMV), geminivirus, brome mosaic virus, and tobacco mosaicvirus.

Generation/Regeneration of Transgenic Plants

Transformed plant cells which are derived by any of the abovetransformation techniques can be cultured to regenerate a whole plantthat possesses the transformed genotype and thus the desired phenotype.Such regeneration techniques rely on manipulation of certainphytohormones in a tissue culture growth medium, typically relying on abiocide and/or herbicide marker which has been introduced together withthe desired nucleotide sequences. Plant regeneration from culturedprotoplasts is described in Payne et al., (1992) Plant Cell and TissueCulture in Liquid Systems John Wiley & Sons, Inc. New York, N.Y.;Gamborg and Phillips (eds) (1995) Plant Cell, Tissue and Organ Culture;Fundamental Methods Springer Lab Manual, Springer-Verlag (BerlinHeidelberg New York); Evans et al. (1983) Protoplasts Isolation andCulture, Handbook of Plant Cell Culture pp. 124-176, MacmillianPublishing Company, New York; and Binding (1985) Regeneration of Plants,Plant Protoplasts pp. 21-73, CRC Press, Boca Raton. Regeneration canalso be obtained from plant callus, explants, somatic embryos (Dandekaret al., (1989) J. Tissue Cult. Meth. 12:145; McGranahan, et al. (1990)Plant Cell Rep. 8:512) organs, or parts thereof. Such regenerationtechniques are described generally in Klee et al., (1987)., Ann. Rev. ofPlant Phys. 38:467-486. Additional details are found in Payne (1992) andJones (1995), both supra, and Weissbach and Weissbach, eds. (1988)Methods for Plant Molecular Biology Academic Press, Inc., San Diego,Calif. This regeneration and growth process includes the steps ofselection of transformant cells and shoots, rooting the transformantshoots and growth of the plantlets in soil. These methods are adapted toproduce transgenic plants bearing QTLs and other genes isolatedaccording to the methods.

In addition, the regeneration of plants containing the polynucleotidesand introduced by Agrobacterium into cells of leaf explants can beachieved as described by Horsch et al., (1985) Science 227:1229-1231. Inthis procedure, transformants are grown in the presence of a selectionagent and in a medium that induces the regeneration of shoots in theplant species being transformed as described by Fraley et al. (1983)Proc. Natl. Acad. Sci. (U.S.A.) 80:4803. This procedure typicallyproduces shoots within two to four weeks and these transformant shootsare then transferred to an appropriate root-inducing medium containingthe selective agent and an antibiotic to prevent bacterial growth.Transgenic plants may be fertile or sterile.

It is not intended that plant transformation and expression ofpolypeptides that provide herbicide tolerance be limited to soybeanspecies. Indeed, it is contemplated that the polypeptides that providetolerance in soybean can also provide a similar phenotype whentransformed and expressed in other plants. Examples of plant genuses andspecies of interest include, but are not limited to, monocots and dicotssuch as corn (Zea mays), Brassica sp. (e.g., B. napus, B. rapa, B.juncea), particularly those Brassica species useful as sources of seedoil, alfalfa (Medicago sativa), rice (Oryza sativa), rye (Secalecereale), sorghum (Sorghum bicolor, Sorghum vulgare), millet (e.g.,pearl millet (Pennisetum glaucum), proso millet (Panicum miliaceum),foxtail millet (Setaria italica), finger millet (Eleusine coracana)),sunflower (Helianthus annuus), safflower (Carthamus tinctorius), wheat(Triticum aestivum), soybean (Glycine max), tobacco (Nicotiana tabacum),potato (Solanum tuberosum), peanuts (Arachis hypogaea), cotton(Gossypium barbadense, Gossypium hirsutum), sweet potato (Ipomoeabatatus), cassava (Manihot esculenta), coffee (Coffea spp.), coconut(Cocos nucifera), pineapple (Ananas comosus), citrus trees (Citrusspp.), cocoa (Theobroma cacao), tea (Camellia sinensis), banana (Musaspp.), avocado (Persea americana), fig (Ficus casica), guava (Psidiumguajava), mango (Mangifera indica), olive (Olea europaea), papaya(Carica papaya), cashew (Anacardium occidentale), macadamia (Macadamiaintegrifolia), almond (Prunus amygdalus), sugar beets (Beta vulgaris),sugarcane (Saccharum spp.), oats (Avena), barley (Hordeum), palm,legumes including beans and peas such as guar, locust bean, fenugreek,garden beans, cowpea, mungbean, lima bean, fava bean, lentils, chickpea,and castor, Arabidopsis, vegetables, ornamentals, grasses, conifers,crop and grain plants that provide seeds of interest, oil-seed plants,and other leguminous plants. Vegetables include tomatoes (Lycopersiconesculentum), lettuce (e.g., Lactuca sativa), green beans (Phaseolusvulgaris), lima beans (Phaseolus limensis), peas (Lathyrus spp.), andmembers of the genus Cucumis such as cucumber (C. sativus), cantaloupe(C. cantalupensis), and musk melon (C. melo). Ornamentals include azalea(Rhododendron spp.), hydrangea (Macrophylla hydrangea), hibiscus(Hibiscus rosasanensis), roses (Rosa spp.), tulips (Tulipa spp.),daffodils (Narcissus spp.), petunias (Petunia hybrida), carnation(Dianthus caryophyllus), poinsettia (Euphorbia pulcherrima), andchrysanthemum. Conifers include, for example, pines such as loblollypine (Pinus taeda), slash pine (Pinus elliotii), ponderosa pine (Pinusponderosa), lodgepole pine (Pinus contorta), and Monterey pine (Pinusradiata); Douglas-fir (Pseudotsuga menziesii); Western hemlock (Tsugacanadensis); Sitka spruce (Picea glauca); redwood (Sequoiasempervirens); true firs such as silver fir (Abies amabilis) and balsamfir (Abies balsamea); and cedars such as Western red cedar (Thujaplicata) and Alaska yellow-cedar (Chamaecyparis nootkatensis).

Promoters of bacterial origin that operate in plants include theoctopine synthase promoter, the nopaline synthase promoter and otherpromoters derived from native Ti plasmids. See, Herrara-Estrella et al.(1983), Nature, 303:209. Viral promoters include the 35S and 19S RNApromoters of cauliflower mosaic virus. See, Odell et al. (1985) Nature,313:810. Other plant promoters include Kunitz trypsin inhibitor promoter(KTI), SCP1, SUP, UCD3, the ribulose-1,3-bisphosphate carboxylase smallsubunit promoter and the phaseolin promoter. The promoter sequence fromthe E8 gene and other genes may also be used. The isolation and sequenceof the E8 promoter is described in detail in Deikman and Fischer (1988)EMBO J. 7:3315. Many other promoters are in current use and can becoupled to an exogenous DNA sequence to direct expression of the nucleicacid.

If expression of a polypeptide from a cDNA is desired, a polyadenylationregion at the 3′-end of the coding region is typically included. Thepolyadenylation region can be derived from the natural gene, from avariety of other plant genes, or from, e.g., T-DNA.

The vector comprising the sequences (e.g., promoters or coding regions)from genes encoding expression products and transgenes will typicallyinclude a nucleic acid subsequence, a marker gene which confers aselectable, or alternatively, a screenable, phenotype on plant cells.For example, the marker can encode biocide tolerance, particularlyantibiotic tolerance, such as tolerance to kanamycin, G418, bleomycin,hygromycin, or herbicide tolerance, such as tolerance to chlorosluforon,or phosphinothricin (the active ingredient in the herbicides bialaphosor Basta). See, e.g., Padgette et al. (1996) In: Herbicide-ResistantCrops (Duke, ed.), pp 53-84, CRC Lewis Publishers, Boca Raton(“Padgette, 1996”). For example, crop selectivity to specific herbicidescan be conferred by engineering genes into crops that encode appropriateherbicide metabolizing enzymes from other organisms, such as microbes.See Vasil (1996) In: Herbicide-Resistant Crops (Duke, ed.), pp 85-91,CRC Lewis Publishers, Boca Raton) (“Vasil”, 1996).

One of skill will recognize that after the recombinant expressioncassette is stably incorporated in transgenic plants and confirmed to beoperable, it can be introduced into other plants by sexual crossing. Anyof a number of standard breeding techniques can be used, depending uponthe species to be crossed. In vegetatively propagated crops, maturetransgenic plants can be propagated by the taking of cuttings or bytissue culture techniques to produce multiple identical plants.Selection of desirable transgenics is made and new varieties areobtained and propagated vegetatively for commercial use. In seedpropagated crops, mature transgenic plants can be self crossed toproduce a homozygous inbred plant. The inbred plant produces seedcontaining the newly introduced heterologous nucleic acid. These seedscan be grown to produce plants that would produce the selectedphenotype. Parts obtained from the regenerated plant, such as flowers,seeds, leaves, branches, fruit, and the like are included, provided thatthese parts comprise cells comprising the isolated nucleic acid. Progenyand variants, and mutants of the regenerated plants are also included,provided that these parts comprise the introduced nucleic acidsequences.

Transgenic or introgressed plants expressing a polynucleotide can bescreened for transmission of the nucleic acid by, for example, standardnucleic acid detection methods or by immunoblot protocols. Expression atthe RNA level can be determined to identify and quantitateexpression-positive plants. Standard techniques for RNA analysis can beemployed and include RT-PCR amplification assays using oligonucleotideprimers designed to amplify only heterologous or introgressed RNAtemplates and solution hybridization assays using marker or linked QTLspecific probes. Plants can also be analyzed for protein expression,e.g., by Western immunoblot analysis using antibodies that recognize theencoded polypeptides. In addition, in situ hybridization andimmunocytochemistry according to standard protocols can be done usingheterologous nucleic acid specific polynucleotide probes and antibodies,respectively, to localize sites of expression within transgenic tissue.Generally, a number of transgenic lines are usually screened for theincorporated nucleic acid to identify and select plants with the mostappropriate expression profiles.

In one example, a transgenic plant that is homozygous for the addedheterologous nucleic acid; e.g., a transgenic plant that contains twoadded nucleic acid sequence copies, e.g., a gene at the same locus oneach chromosome of a homologous chromosome pair is provided. Ahomozygous transgenic plant can be obtained by sexually mating(self-fertilizing) a heterozygous transgenic plant that contains asingle added heterologous nucleic acid, germinating some of the seedproduced and analyzing the resulting plants produced for alteredexpression of a polynucleotide relative to a control plant (e.g., anative, non-transgenic plant). Back-crossing to a parental plant andout-crossing with a non-transgenic plant can be used to introgress theheterologous nucleic acid into a selected background (e.g., an elite orexotic soybean line).

Stacking of Traits and Additional Traits of Interest

In some embodiments, the polynucleotide conferring the tolerance in theplants are engineered into a molecular stack with at least oneadditional polynucleotide. The additional polynucleotide may confer anyadditional trait of interest, such as tolerance to an additionalherbicide, insects, disease, or any other desirable trait. A trait, asused herein, refers to the phenotype derived from a particular sequenceor groups of sequences. For example, herbicide-tolerance polynucleotidesmay be stacked with any other polynucleotides encoding polypeptideshaving pesticidal and/or insecticidal activity, such as Bacillusthuringiensis toxic proteins (described in U.S. Pat. Nos. 5,366,892;5,747,450; 5,737,514; 5,723,756; 5,593,881; Geiser et al. (1986) Gene48: 109; Lee et al. (2003) Appl. Environ. Microbiol. 69: 4648-4657(Vip3A); Galitzky et al. (2001) Acta Crystallogr. D. Biol. Crystallogr.57:1101-1109 (Cry3Bb1); and Herman et al. (2004) J. Agric. Food Chem.52: 2726-2734 (Cry1F)), lectins (Van Damme et al. (1994) Plant Mol.Biol. 24: 825, pentin (described in U.S. Pat. No. 5,981,722), and thelike. The combinations generated can also include multiple copies of anyone of the polynucleotides of interest.

In some embodiments, herbicide-tolerance polynucleotide may be stackedwith other herbicide-tolerance traits to create a transgenic plant withfurther improved properties. Other herbicide-tolerance polynucleotidesthat could be used in such embodiments include those conferringtolerance to the same herbicide by other modes of action, or a differentherbicide. Other traits that could be combined with herbicide-tolerancepolynucleotides include those derived from polynucleotides that conferon the plant the capacity to produce a higher level of5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), for example, asmore fully described in U.S. Pat. Nos. 6,248,876 B1; 5,627,061;5,804,425; 5,633,435; 5,145,783; 4,971,908; 5,312,910; 5,188,642;4,940,835; 5,866,775; 6,225,114 B1; 6,130,366; 5,310,667; 4,535,060;4,769,061; 5,633,448; 5,510,471; U.S. Pat. No. Re. 36,449; U.S. Pat.Nos. RE 37,287 E; and 5,491,288; and WO 97/04103; WO 00/66746; WO01/66704; and WO 00/66747. Other traits that could be combined withherbicide-tolerance polynucleotides include those conferring toleranceto sulfonylurea and/or imidazolinone, for example, as described morefully in U.S. Pat. Nos. 5,605,011; 5,013,659; 5,141,870; 5,767,361;5,731,180; 5,304,732; 4,761,373; 5,331,107; 5,928,937; and 5,378,824;and international publication WO 96/33270.

In some embodiments, herbicide-tolerance polynucleotides of the plantsmay be stacked with, for example, hydroxyphenylpyruvatedioxygenaseswhich are enzymes that catalyze the reaction in whichpara-hydroxyphenylpyruvate (HPP) is transformed into homogentisate.Molecules which inhibit this enzyme and which bind to the enzyme inorder to inhibit transformation of the HPP into homogentisate are usefulas herbicides. Traits conferring tolerance to such herbicides in plantsare described in U.S. Pat. Nos. 6,245,968 B1; 6,268,549; and 6,069,115;and WO 99/23886. Other examples of suitable herbicide-tolerance traitsthat could be stacked with herbicide-tolerance polynucleotides includearyloxyalkanoate dioxygenase polynucleotides (which reportedly confertolerance to 2,4-D and other phenoxy auxin herbicides as well as toaryloxyphenoxypropionate herbicides as described, for example, inWO2005/107437) and dicamba-tolerance polynucleotides as described, forexample, in Herman et al. (2005) J. Biol. Chem. 280: 24759-24767.

Other examples of herbicide-tolerance traits that could be combined withherbicide-tolerance polynucleotides include those conferred bypolynucleotides encoding an exogenous phosphinothricinacetyltransferase, as described in U.S. Pat. Nos. 5,969,213; 5,489,520;5,550,318; 5,874,265; 5,919,675; 5,561,236; 5,648,477; 5,646,024;6,177,616; and 5,879,903. Plants containing an exogenousphosphinothricin acetyltransferase can exhibit improved tolerance toglufosinate herbicides, which inhibit the enzyme glutamine synthase.Other examples of herbicide-tolerance traits that could be combined withthe herbicide-tolerance polynucleotides include those conferred bypolynucleotides conferring altered protoporphyrinogen oxidase (protox)activity, as described in U.S. Pat. Nos. 6,288,306 B1; 6,282,837 B1; and5,767,373; and WO 01/12825. Plants containing such polynucleotides canexhibit improved tolerance to any of a variety of herbicides whichtarget the protox enzyme (also referred to as “protox inhibitors”).

Other examples of herbicide-tolerance traits that could be combined withherbicide-tolerance polynucleotides include those conferring toleranceto at least one herbicide in a plant such as, for example, a maize plantor horseweed. Herbicide-tolerant weeds are known in the art, as areplants that vary in their tolerance to particular herbicides. See, e.g.,Green and Williams (2004) “Correlation of Corn (Zea mays) InbredResponse to Nicosulfuron and Mesotrione,” poster presented at the WSSAAnnual Meeting in Kansas City, Mo., Feb. 9-12, 2004; Green (1998) WeedTechnology 12: 474-477; Green and Ulrich (1993) Weed Science 41:508-516. The trait(s) responsible for these tolerances can be combinedby breeding or via other methods with herbicide-tolerancepolynucleotides to provide a plant as well as methods of use thereof.

In this manner, plants that are more tolerant to multiple herbicides aredisclosed. Accordingly, methods for growing a crop (i.e., forselectively controlling weeds in an area of cultivation) that comprisetreating an area of interest (e.g., a field or area of cultivation) withat least one herbicide to which the plant is tolerant are likewisedisclosed. In some embodiments, methods further comprise treatment withadditional herbicides to which the plant is tolerant. In suchembodiments, generally the methods permit selective control of weedswithout significantly damaging the crop. As used herein, an “area ofcultivation” comprises any region in which one desires to grow a plant.Such areas of cultivations include, but are not limited to, a field inwhich a plant is cultivated (such as a crop field, a sod field, a treefield, a managed forest, a field for culturing fruits and vegetables,etc), a greenhouse, a growth chamber, etc.

Herbicide-tolerant traits can also be combined with at least one othertrait to produce plants that further comprise a variety of desired traitcombinations including, but not limited to, traits desirable for animalfeed such as high oil content (e.g., U.S. Pat. No. 6,232,529); increaseddigestibility (e.g., modified storage proteins (U.S. application Ser.No. 10/053,410, filed Nov. 7, 2001); and thioredoxins (U.S. applicationSer. No. 10/005,429, filed Dec. 3, 2001)); the disclosures of which areherein incorporated by reference. Desired trait combinations alsoinclude LLNC (low linolenic acid content; see, e.g., Dyer et al. (2002)Appl. Microbiol. Biotechnol. 59: 224-230) and OLCH (high oleic acidcontent; see, e.g., Fernandez-Moya et al. (2005) J. Agric. Food Chem.53: 5326-5330).

Herbicide-tolerant traits of interest can also be combined with otherdesirable traits such as, for example, fumonisim detoxification genes(U.S. Pat. No. 5,792,931), avirulence and disease resistance genes(Jones et al. (1994) Science 266: 789; Martin et al. (1993) Science 262:1432; Mindrinos et al. (1994) Cell 78: 1089), and traits desirable forprocessing or process products such as modified oils (e.g., fatty aciddesaturase genes (U.S. Pat. No. 5,952,544; WO 94/11516)); modifiedstarches (e.g., ADPG pyrophosphorylases (AGPase), starch synthases (SS),starch branching enzymes (SBE), and starch debranching enzymes (SDBE));and polymers or bioplastics (e.g., U.S. Pat. No. 5,602,321;beta-ketothiolase, polyhydroxybutyrate synthase, and acetoacetyl-CoAreductase (Schubert et al. (1988) J. Bacteriol. 170:5837-5847)facilitate expression of polyhydroxyalkanoates (PHAs)); the disclosuresof which are herein incorporated by reference. One could also combineherbicide-tolerant polynucleotides with polynucleotides providingagronomic traits such as male sterility (e.g., see U.S. Pat. No.5,583,210), stalk strength, flowering time, or transformation technologytraits such as cell cycle regulation or gene targeting (e.g., WO99/61619, WO 00/17364, and WO 99/25821); the disclosures of which areherein incorporated by reference.

In another embodiment, the herbicide-tolerant traits of interest canalso be combined with the Rcg1 sequence or biologically active variantor fragment thereof. The Rcg1 sequence is an anthracnose stalk rotresistance gene in corn. See, e.g., U.S. patent application Ser. Nos.11/397,153, 11/397,275, and 11/397,247, each of which is hereinincorporated by reference.

These stacked combinations can be created by any method including, butnot limited to, breeding plants by any conventional or TopCrossmethodology, or genetic transformation. If the sequences are stacked bygenetically transforming the plants, the polynucleotide sequences ofinterest can be combined at any time and in any order. For example, atransgenic plant comprising one or more desired traits can be used asthe target to introduce further traits by subsequent transformation. Thetraits can be introduced simultaneously in a co-transformation protocolwith the polynucleotides of interest provided by any combination oftransformation cassettes. For example, if two sequences will beintroduced, the two sequences can be contained in separatetransformation cassettes (trans) or contained on the same transformationcassette (cis). Expression of the sequences can be driven by the samepromoter or by different promoters. In certain cases, it may bedesirable to introduce a transformation cassette that will suppress theexpression of the polynucleotide of interest. This may be combined withany combination of other suppression cassettes or overexpressioncassettes to generate the desired combination of traits in the plant. Itis further recognized that polynucleotide sequences can be stacked at adesired genomic location using a site-specific recombination system.See, e.g., WO99/25821, WO99/25854, WO99/25840, WO99/25855, andWO99/25853, all of which are herein incorporated by reference.

Insect resistance genes may encode resistance to pests that have greatyield drag such as rootworm, cutworm, European Corn Borer, and the like.Such genes include, for example, Bacillus thuringiensis toxic proteingenes (U.S. Pat. Nos. 5,366,892; 5,747,450; 5,736,514; 5,723,756;5,593,881; and Geiser et al. (1986) Gene 48: 109); and the like.

Genes encoding disease resistance traits include detoxification genes,such as against fumonisin (U.S. Pat. No. 5,792,931); avirulence (avr)and disease resistance (R) genes (Jones et al. (1994) Science 266: 789;Martin et al. (1993) Science 262: 1432; and Mindrinos et al. (1994) Cell78: 1089); and the like.

Sterility genes can also be encoded in an expression cassette andprovide an alternative to physical detasseling. Examples of genes usedin such ways include male tissue-preferred genes and genes with malesterility phenotypes such as QM, described in U.S. Pat. No. 5,583,210.Other genes include kinases and those encoding compounds toxic to eithermale or female gametophytic development.

Polynucleotide Constructs

In specific embodiments, one or more of the herbicide-tolerantpolynucleotides employed in the methods and compositions can be providedin an expression cassette for expression in the plant or other organismof interest. The cassette will include 5′ and 3′ regulatory sequencesoperably linked to a herbicide-tolerance polynucleotide. “Operablylinked” is intended to mean a functional linkage between two or moreelements. For example, an operable linkage between a polynucleotide ofinterest and a regulatory sequence (e.g., a promoter) is functional linkthat allows for expression of the polynucleotide of interest. Operablylinked elements may be contiguous or non-contiguous. When used to referto the joining of two protein coding regions, by “operably linked” isintended that the coding regions are in the same reading frame. Whenused to refer to the effect of an enhancer, “operably linked” indicatesthat the enhancer increases the expression of a particularpolynucleotide or polynucleotides of interest. Where the polynucleotideor polynucleotides of interest encode a polypeptide, the encodedpolypeptide is produced at a higher level.

The cassette may additionally contain at least one additional gene to becotransformed into the organism. Alternatively, the additional gene(s)can be provided on multiple expression cassettes. Such an expressioncassette is provided with a plurality of restriction sites and/orrecombination sites for insertion of the herbicide-tolerancepolynucleotide to be under the transcriptional regulation of theregulatory regions. The expression cassette may additionally containother genes, including other selectable marker genes. Where a cassettecontains more than one polynucleotide, the polynucleotides in thecassette may be transcribed in the same direction or in differentdirections (also called “divergent” transcription).

An expression cassette comprising a herbicide-tolerance polynucleotidewill include in the 5′-3′ direction of transcription a transcriptionaland translational initiation region (i.e., a promoter), aherbicide-tolerance polynucleotide, and a transcriptional andtranslational termination region (i.e., termination region) functionalin plants or the other organism of interest. Accordingly, plants havingsuch expression cassettes are also provided. The regulatory regions(i.e., promoters, transcriptional regulatory regions, and translationaltermination regions) and/or the herbicide-tolerance polynucleotide maybe native (i.e., analogous) to the host cell or to each other.Alternatively, the regulatory regions and/or the herbicide-tolerancepolynucleotide may be heterologous to the host cell or to each other.

While it may be optimal to express polynucleotides using heterologouspromoters, native promoter sequences may be used. Such constructs canchange expression levels and/or expression patterns of the encodedpolypeptide in the plant or plant cell. Expression levels and/orexpression patterns of the encoded polypeptide may also be changed as aresult of an additional regulatory element that is part of theconstruct, such as, for example, an enhancer. Thus, the phenotype of theplant or cell can be altered even though a native promoter is used.

The termination region may be native with the transcriptional initiationregion, may be native with the operably linked herbicide-tolerancepolynucleotide of interest, may be native with the plant host, or may bederived from another source (i.e., foreign or heterologous) to thepromoter, the herbicide-tolerance polynucleotide of interest, the planthost, or any combination thereof. Convenient termination regions areavailable from the Ti-plasmid of A. tumefaciens, such as the octopinesynthase and nopaline synthase termination regions, or can be obtainedfrom plant genes such as the Solanum tuberosum proteinase inhibitor IIgene. See Guerineau et al. (1991) Mol. Gen. Genet. 262: 141-144;Proudfoot (1991) Cell 64: 671-674; Sanfacon et al. (1991) Genes Dev. 5:141-149; Mogen et al. (1990) Plant Cell 2: 1261-1272; Munroe et al.(1990) Gene 91: 151-158; Ballas et al. (1989) Nucleic Acids Res. 17:7891-7903; and Joshi et al. (1987) Nucleic Acids Res. 15: 9627-9639.

A number of promoters can be used, including the native promoter of thepolynucleotide sequence of interest. The promoters can be selected basedon the desired outcome. The polynucleotides of interest can be combinedwith constitutive, tissue-preferred, or other promoters for expressionin plants.

Such constitutive promoters include, for example, the core promoter ofthe Rsyn7 promoter and other constitutive promoters disclosed in WO99/43838 and U.S. Pat. No. 6,072,050; the core CaMV 35S promoter (Odellet al. (1985) Nature 313: 810-812); rice actin (McElroy et al. (1990)Plant Cell 2: 163-171); the maize actin promoter; the ubiquitin promoter(see, e.g., Christensen et al. (1989) Plant Mol. Biol. 12: 619-632;Christensen et al. (1992) Plant Mol. Biol. 18: 675-689; Callis et al.(1995) Genetics 139: 921-39); pEMU (Last et al. (1991) Theor. Appl.Genet. 81: 581-588); MAS (Velten et al. (1984) EMBO J. 3: 2723-2730);ALS promoter (U.S. Pat. No. 5,659,026), and the like. Other constitutivepromoters include, for example, those described in U.S. Pat. Nos.5,608,149; 5,608,144; 5,604,121; 5,569,597; 5,466,785; 5,399,680;5,268,463; 5,608,142; and 6,177,611. Some promoters show improvedexpression when they are used in conjunction with a native 5′untranslated region and/or other elements such as, for example, anintron. For example, the maize ubiquitin promoter is often placedupstream of a polynucleotide of interest along with at least a portionof the 5′ untranslated region of the ubiquitin gene, including the firstintron of the maize ubiquitin gene.

Chemical-regulated promoters can be used to modulate the expression of agene in a plant through the application of an exogenous chemicalregulator. Depending upon the objective, the promoter may be achemical-inducible promoter for which application of the chemicalinduces gene expression or the promoter may be a chemical-repressiblepromoter for which application of the chemical represses geneexpression. Chemical-inducible promoters are known in the art andinclude, but are not limited to, the maize In2-2 promoter, which isactivated by benzenesulfonamide herbicide safeners, the maize GSTpromoter, which is activated by hydrophobic electrophilic compounds thatare used as pre-emergent herbicides, and the tobacco PR-1a promoter,which is activated by salicylic acid. Other chemical-regulated promotersof interest include steroid-responsive promoters (see, e.g., theglucocorticoid-inducible promoter in Schena et al. (1991) Proc. Natl.Acad. Sci. USA 88: 10421-10425 and McNellis et al. (1998) Plant J.14(2): 247-257) and tetracycline-inducible and tetracycline-repressiblepromoters (see, e.g., Gatz et al. (1991) Mol. Gen. Genet. 227: 229-237,and U.S. Pat. Nos. 5,814,618 and 5,789,156), herein incorporated byreference).

Tissue-preferred promoters can be utilized to target enhancedherbicide-tolerance polypeptide expression within a particular planttissue. Tissue-preferred promoters include Yamamoto et al. (1997) PlantJ. 12: 255-265; Kawamata et al. (1997) Plant Cell Physiol. 38: 792-803;Hansen et al. (1997) Mol. Gen Genet. 254: 337-343; Russell et al. (1997)Transgenic Res. 6: 157-168; Rinehart et al. (1996) Plant Physiol.112:1331-1341; Van Camp et al. (1996) Plant Physiol. 112: 525-535;Canevascini et al. (1996) Plant Physiol. 112: 513-524; Yamamoto et al.(1994) Plant Cell Physiol. 35: 773-778; Lam (1994) Results Probl. CellDiffer. 20: 181-196; Orozco et al. (1993) Plant Mol Biol. 23: 1129-1138;Matsuoka et al. (1993) Proc Natl. Acad. Sci. USA 90: 9586-9590; andGuevara-Garcia et al. (1993) Plant J. 4: 495-505. Such promoters can bemodified, if necessary, for weak expression.

Leaf-preferred promoters are known in the art. See, e.g., Yamamoto etal. (1997) Plant J. 12: 255-265; Kwon et al. (1994) Plant Physiol. 105:357-67; Yamamoto et al. (1994) Plant Cell Physiol. 35: 773-778; Gotor etal. (1993) Plant J. 3: 509-18; Orozco et al. (1993) Plant Mol. Biol. 23:1129-1138; and Matsuoka et al. (1993) Proc. Natl. Acad. Sci. USA 90:9586-9590.

Root-preferred promoters are known and can be selected from the manyavailable from the literature or isolated de novo from variouscompatible species. See, e.g., Hire et al. (1992) Plant Mol. Biol.20(2): 207-218 (soybean root-specific glutamine synthetase gene); Kellerand Baumgartner (1991) Plant Cell 3: 1051-1061 (root-specific controlelement in the GRP 1.8 gene of French bean); Sanger et al. (1990) PlantMol. Biol. 14: 433-443 (root-specific promoter of the mannopine synthase(MAS) gene of Agrobacterium tumefaciens); and Miao et al. (1991) PlantCell 3: 11-22 (full-length cDNA clone encoding cytosolic glutaminesynthetase (GS), which is expressed in roots and root nodules ofsoybean). See also Bogusz et al. (1990) Plant Cell 2: 633-641, where tworoot-specific promoters are described. Leach and Aoyagi (1991) describetheir analysis of the promoters of the highly expressed rolC and rolDroot-inducing genes of Agrobacterium rhizogenes (see Plant Science(Limerick) 79: 69-76). They concluded that enhancer and tissue-preferredDNA determinants are dissociated in those promoters. Teeri et al. (1989)used gene fusion to lacZ to show that the Agrobacterium T-DNA geneencoding octopine synthase is especially active in the epidermis of theroot tip and that the TR2′ gene is root specific in the intact plant andstimulated by wounding in leaf tissue, an especially desirablecombination of characteristics for use with an insecticidal orlarvacidal gene (see EMBO J. 8: 343-350). The TR1′ gene, fused to nptII(neomycin phosphotransferase II) showed similar characteristics.Additional root-preferred promoters include the VfENOD-GRP3 genepromoter (Kuster et al. (1995) Plant Mol. Biol. 29: 759-772); and rolBpromoter (Capana et al. (1994) Plant Mol. Biol. 25: 681-691. See alsoU.S. Pat. Nos. 5,837,876; 5,750,386; 5,633,363; 5,459,252; 5,401,836;5,110,732; and 5,023,179.

Seed-preferred promoters include both seed-specific promoters (thosepromoters active during seed development such as promoters of seedstorage proteins) as well as “seed-germinating” promoters (thosepromoters active during seed germination). See Thompson et al. (1989)BioEssays 10: 108, herein incorporated by reference. Such seed-preferredpromoters include, but are not limited to, Cim1 (cytokinin-inducedmessage); cZ19B1 (maize 19 kDa zein); milps (myo-inositol-1-phosphatesynthase) (see WO 00/11177 and U.S. Pat. No. 6,225,529; hereinincorporated by reference). Gamma-zein is an endosperm-specificpromoter. Globulin 1 (Glb-1) is a representative embryo-specificpromoter. For dicots, seed-specific promoters include, but are notlimited to, bean .beta.-phaseolin, napin, .beta.-conglycinin, soybeanlectin, cruciferin, and the like. For monocots, seed-specific promotersinclude, but are not limited to, maize 15 kDa zein, 22 kDa zein, 27 kDazein, gamma-zein, waxy, shrunken 1, shrunken 2, Globulin 1, etc. Seealso WO 00/12733, where seed-preferred promoters from end1 and end2genes are disclosed; herein incorporated by reference.

Additional promoters of interest include the SCP1 promoter (U.S. Pat.No. 6,072,050), the HB2 promoter (U.S. Pat. No. 6,177,611) and the SAMSpromoter (US20030226166 and SEQ ID NO: 87 and biologically activevariants and fragments thereof); each of which is herein incorporated byreference. In addition, as discussed elsewhere herein, various enhancerscan be used with these promoters including, for example, the ubiquitinintron (i.e, the maize ubiquitin intron 1 (see, e.g., NCBI sequenceS94464), the omega enhancer or the omega prime enhancer (Gallie et al.(1989) Molecular Biology of RNA ed. Cech (Liss, N.Y.) 237-256 and Gallieet al. Gene (1987) 60:217-25), or the 35S enhancer; each of which isincorporated by reference.

The expression cassette can also comprise a selectable marker gene forthe selection of transformed cells. Selectable marker genes are utilizedfor the selection of transformed cells or tissues. Marker genes includegenes encoding antibiotic resistance, such as those encoding neomycinphosphotransferase II (NEO) and hygromycin phosphotransferase (HPT), aswell as genes conferring resistance to herbicidal compounds, such asglufosinate ammonium, bromoxynil, imidazolinones, and2,4-dichlorophenoxyacetate (2,4-D). Additional selectable markersinclude phenotypic markers such as .beta.-galactosidase and fluorescentproteins such as green fluorescent protein (GFP) (Su et al. (2004)Biotechnol Bioeng 85: 610-9 and Fetter et al. (2004) Plant Cell 16:215-28), cyan florescent protein (CYP) (Bolte et al. (2004) J. CellScience 117: 943-54 and Kato et al. (2002) Plant Physiol 129: 913-42),and yellow fluorescent protein (PhiYFP from Evrogen, see, Bolte et al.(2004) J. Cell Science 117: 943-54). For additional selectable markers,see generally Yarranton (1992) Curr. Opin. Biotech. 3: 506-511;Christopherson et al. (1992) Proc. Natl. Acad. Sci. USA 89: 6314-6318;Yao et al. (1992) Cell 71: 63-72; Reznikoff (1992) Mol. Microbiol. 6:2419-2422; Barkley et al. (1980) in The Operon, pp. 177-220; Hu et al.(1987) Cell 48: 555-566; Brown et al. (1987) Cell 49: 603-612; Figge etal. (1988) Cell 52: 713-722; Deuschle et al. (1989) Proc. Natl. Acad.Aci. USA 86: 5400-5404; Fuerst et al. (1989) Proc. Natl. Acad. Sci. USA86: 2549-2553; Deuschle et al. (1990) Science 248: 480-483; Gossen(1993) Ph.D. Thesis, University of Heidelberg; Reines et al. (1993)Proc. Natl. Acad. Sci. USA 90: 1917-1921; Labow et al. (1990) Mol. Cell.Biol. 10: 3343-3356; Zambretti et al. (1992) Proc. Natl. Acad. Sci. USA89: 3952-3956; Baim et al. (1991) Proc. Natl. Acad. Sci. USA 88:5072-5076; Wyborski et al. (1991) Nucleic Acids Res. 19: 4647-4653;Hillenand-Wissman (1989) Topics Mol. Struc. Biol. 10: 143-162; Degenkolbet al. (1991) Antimicrob. Agents Chemother. 35: 1591-1595; Kleinschmidtet al. (1988) Biochemistry 27: 1094-1104; Bonin (1993) Ph.D. Thesis,University of Heidelberg; Gossen et al. (1992) Proc. Natl. Acad. Sci.USA 89: 5547-5551; Oliva et al. (1992) Antimicrob. Agents Chemother. 36:913-919; Hlavka et al. (1985) Handbook of Experimental Pharmacology,Vol. 78 (Springer-Verlag, Berlin); Gill et al. (1988) Nature 334:721-724. Such disclosures are herein incorporated by reference. Theabove list of selectable marker genes is not meant to be limiting. Anyselectable marker gene can be used, including the GAT gene and/or HRAgene.

Methods are known in the art of increasing the expression level of apolypeptide in a plant or plant cell, for example, by inserting into thepolypeptide coding sequence one or two G/C-rich codons (such as GCG orGCT) immediately adjacent to and downstream of the initiating methionineATG codon. Where appropriate, the polynucleotides may be modified forincreased expression in the transformed plant. That is, thepolynucleotides can be synthesized substituting in the polypeptidecoding sequence one or more codons which are less frequently utilized inplants for codons encoding the same amino acid(s) which are morefrequently utilized in plants, and introducing the modified codingsequence into a plant or plant cell and expressing the modified codingsequence. See, e.g., Campbell and Gowri (1990) Plant Physiol. 92: 1-11for a discussion of host-preferred codon usage. Methods are available inthe art for synthesizing plant-preferred genes. See, e.g., U.S. Pat.Nos. 5,380,831, and 5,436,391, and Murray et al. (1989) Nucleic AcidsRes. 17: 477-498, herein incorporated by reference. Embodimentscomprising such modifications are also a feature disclosed.

Additional sequence modifications are known to enhance gene expressionin a cellular host. These include elimination of sequences encodingspurious polyadenylation signals, exon-intron splice site signals,transposon-like repeats, and other such well-characterized sequencesthat may be deleterious to gene expression. The G-C content of thesequence may be adjusted to levels average for a given cellular host, ascalculated by reference to known genes expressed in the host cell. Whenpossible, the sequence is modified to avoid predicted hairpin secondarymRNA structures. “Enhancers” such as the CaMV 35S enhancer may also beused (see, e.g., Benfey et al. (1990) EMBO J. 9: 1685-96), or otherenhancers may be used. For example, the sequence set forth in SEQ ID NO:1, 72, 79, 84, 85, 88, or 89 or a biologically active variant orfragment thereof can be used. See also U.S. Utility application Ser. No.11/508,045, entitled “Methods and Compositions for the Expression of aPolynucleotide of Interest.” As used herein, an enhancer, when operablylinked to an appropriate promoter, will modulate the level oftranscription of an operably linked polynucleotide of interest.Biologically active fragments and variants of the enhancer domain mayretain the biological activity of modulating (increase or decrease) thelevel of transcription when operably linked to an appropriate promoter.

Generally, variants of a particular polynucleotide will have at leastabout 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to anotherpolynucleotide as determined by sequence alignment programs andparameters. Variants of a particular polynucleotides also include thoseencoding a polypeptide having at least about 40%, 45%, 50%, 55%, 60%,65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99% or more sequence identity to a reference polypeptide as determinedby sequence alignment programs and parameters. Polypeptide variantsinclude those encoded by variant polynucleotides, and those having atleast about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to areference polypeptide as determined by sequence alignment programs andparameters.

The expression cassette may additionally contain 5′ leader sequences.Such leader sequences can act to enhance translation. Translationleaders are known in the art and include: picornavirus leaders, forexample, EMCV leader (Encephalomyocarditis 5′ noncoding region)(Elroy-Stein et al. (1989) Proc. Natl. Acad. Sci. USA 86: 6126-6130);potyvirus leaders, for example, TEV leader (Tobacco Etch Virus) (Gallieet al. (1995) Gene 165(2): 233-238), MDMV leader (Maize Dwarf MosaicVirus) (Virology 154: 9-20), and human immunoglobulin heavy-chainbinding protein (BiP) (Macejak et al. (1991) Nature 353: 90-94);untranslated leader from the coat protein mRNA of alfalfa mosaic virus(AMV RNA 4) (Jobling et al. (1987) Nature 325: 622-625); tobacco mosaicvirus leader (TMV) (Gallie et al. (1989) in Molecular Biology of RNA,ed. Cech (Liss, N.Y.), pp. 237-256); and maize chlorotic mottle virusleader (MCMV) (Lommel et al. (1991) Virology 81: 382-385). See also,Della-Cioppa et al. (1987) Plant Physiol. 84: 965-968.

In preparing the expression cassette, the various polynucleotidefragments may be manipulated, so as to provide for sequences to be inthe proper orientation and, as appropriate, in the proper reading frame.Toward this end, adapters or linkers may be employed to join thefragments or other manipulations may be involved to provide forconvenient restriction sites, removal of superfluous material such asthe removal of restriction sites, or the like. For this purpose, invitro mutagenesis, primer repair, restriction, annealing,resubstitutions, e.g., transitions and transversions, may be involved.Standard recombinant DNA and molecular cloning techniques used hereinare well known in the art and are described more fully, for example, inSambrook et al. (1989) Molecular Cloning: A Laboratory Manual (ColdSpring Harbor Laboratory Press, Cold Spring Harbor) (also known as“Maniatis”).

In some embodiments, the polynucleotide of interest is targeted to thechloroplast for expression. In this manner, where the polynucleotide ofinterest is not directly inserted into the chloroplast, the expressioncassette will additionally contain a nucleic acid encoding a transitpeptide to direct the gene product of interest to the chloroplasts. Suchtransit peptides are known in the art. See, e.g., Von Heijne et al.(1991) Plant Mol. Biol. Rep. 9: 104-126; Clark et al. (1989) J. Biol.Chem. 264: 17544-17550; Della-Cioppa et al. (1987) Plant Physiol. 84:965-968; Romer et al. (1993) Biochem. Biophys. Res. Commun. 196:1414-1421; and Shah et al. (1986) Science 233: 478-481.

Chloroplast targeting sequences are known in the art and include thechloroplast small subunit of ribulose-1,5-bisphosphate carboxylase(Rubisco) (de Castro Silva Filho et al. (1996) Plant Mol. Biol. 30:769-780; Schnell et al. (1991) J. Biol. Chem. 266(5): 3335-3342);5-(enolpyruvyl)shikimate-3-phosphate synthase (EPSPS) (Archer et al.(1990) J. Bioenerg Biomemb. 22(6): 789-810); tryptophan synthase (Zhaoet al. (1995) J. Biol. Chem. 270(11): 6081-6087); plastocyanin (Lawrenceet al. (1997) J. Biol. Chem. 272(33): 20357-20363); chorismate synthase(Schmidt et al. (1993) J. Biol. Chem. 268(36): 27447-27457); and thelight harvesting chlorophyll a/b binding protein (LHBP) (Lamppa et al.(1988) J. Biol. Chem. 263: 14996-14999). See also Von Heijne et al.(1991) Plant Mol. Biol. Rep. 9: 104-126; Clark et al. (1989) J. Biol.Chem. 264: 17544-17550; Della-Cioppa et al. (1987) Plant Physiol. 84:965-968; Romer et al. (1993) Biochem. Biophys. Res. Commun. 196:1414-1421; and Shah et al. (1986) Science 233: 478-481.

Methods for transformation of chloroplasts are known in the art. See,e.g., Svab et al. (1990) Proc. Natl. Acad. Sci. USA 87: 8526-8530; Svaband Maliga (1993) Proc. Natl. Acad. Sci. USA 90: 913-917; Svab andMaliga (1993) EMBO J. 12: 601-606. The method relies on particle gundelivery of DNA containing a selectable marker and targeting of the DNAto the plastid genome through homologous recombination. Additionally,plastid transformation can be accomplished by transactivation of asilent plastid-borne transgene by tissue-preferred expression of anuclear-encoded and plastid-directed RNA polymerase. Such a system hasbeen reported in McBride et al. (1994) Proc. Natl. Acad. Sci. USA 91:7301-7305.

The polynucleotides of interest to be targeted to the chloroplast may beoptimized for expression in the chloroplast to account for differencesin codon usage between the plant nucleus and this organelle. In thismanner, the polynucleotide of interest may be synthesized usingchloroplast-preferred codons. See, e.g., U.S. Pat. No. 5,380,831, hereinincorporated by reference.

Methods of Introducing

Compositions include plants generated by introducing a polypeptide orpolynucleotide into a plant. “Introducing” is intended to meanpresenting to the plant the polynucleotide or polypeptide in such amanner that the sequence gains access to the interior of a cell of theplant. The methods do not depend on a particular method for introducinga sequence into a plant, only that the polynucleotide or polypeptidesgains access to the interior of at least one cell of the plant. Methodsfor introducing polynucleotide or polypeptides into plants are known inthe art including, but not limited to, stable transformation methods,transient transformation methods, virus-mediated methods, and breeding.

“Stable transformation” is intended to mean that the nucleotideconstruct introduced into a plant integrates into the genome of theplant and is capable of being inherited by the progeny thereof“Transient transformation” is intended to mean that a polynucleotide isintroduced into the plant and does not integrate into the genome of theplant or a polypeptide is introduced into a plant.

Transformation protocols as well as protocols for introducingpolypeptides or polynucleotide sequences into plants may vary dependingon the type of plant or plant cell (i.e., monocot or dicot) targeted fortransformation. Suitable methods of introducing polypeptides andpolynucleotides into plant cells include microinjection (Crossway et al.(1986) Biotechniques 4: 320-334), electroporation (Riggs et al. (1986)Proc. Natl. Acad. Sci. USA 83: 5602-5606, Agrobacterium-mediatedtransformation (U.S. Pat. No. 5,563,055 and U.S. Pat. No. 5,981,840),direct gene transfer (Paszkowski et al. (1984) EMBO J. 3: 2717-2722),and ballistic particle acceleration (see, for example, U.S. Pat. No.4,945,050; U.S. Pat. No. 5,879,918; U.S. Pat. Nos. 5,886,244; and,5,932,782; Tomes et al. (1995) in Plant Cell, Tissue, and Organ Culture:Fundamental Methods, ed. Gamborg and Phillips (Springer-Verlag, Berlin);McCabe et al. (1988) Biotechnology 6: 923-926); and Lec1 transformation(WO 00/28058). Also see Weissinger et al. (1988) Ann. Rev. Genet. 22:421-477; Sanford et al. (1987) Particulate Science and Technology 5:27-37 (onion); Christou et al. (1988) Plant Physiol. 87: 671-674(soybean); McCabe et al. (1988) Bio/Technology 6: 923-926 (soybean);Finer and McMullen (1991) In Vitro Cell Dev. Biol. 27P: 175-182(soybean); Singh et al. (1998) Theor. Appl. Genet. 96:319-324 (soybean);Datta et al. (1990) Biotechnology 8: 736-740 (rice); Klein et al. (1988)Proc. Natl. Acad. Sci. USA 85:4305-4309 (maize); Klein et al. (1988)Biotechnology 6:559-563 (maize); U.S. Pat. Nos. 5,240,855; 5,322,783;and, 5,324,646; Klein et al. (1988) Plant Physiol. 91: 440-444 (maize);Fromm et al. (1990) Biotechnology 8: 833-839 (maize); protocolspublished electronically by “IP.com” under the permanent publicationidentifiers IPCOM000033402D, IPCOM000033402D, and IPCOM000033402D andavailable at the “IP.com” website (cotton); Hooykaas-Van Slogteren etal. (1984) Nature (London) 311: 763-764; U.S. Pat. No. 5,736,369(cereals); Bytebier et al. (1987) Proc. Natl. Acad. Sci. USA 84:5345-5349 (Liliaceae); De Wet et al. (1985) in The ExperimentalManipulation of Ovule Tissues, ed. Chapman et al. (Longman, N.Y.), pp.197-209 (pollen); Kaeppler et al. (1990) Plant Cell Reports 9: 415-418and Kaeppler et al. (1992) Theor. Appl. Genet. 84: 560-566(whisker-mediated transformation); D'Halluin et al. (1992) Plant Cell 4:1495-1505 (electroporation); Li et al. (1993) Plant Cell Reports 12:250-255 and Christou and Ford (1995) Annals of Botany 75: 407-413(rice); Osjoda et al. (1996) Nature Biotechnology 14: 745-750 (maize viaAgrobacterium tumefaciens); all of which are herein incorporated byreference.

In specific embodiments, herbicide-tolerance or other desirablesequences can be provided to a plant using a variety of transienttransformation methods. Such transient transformation methods include,but are not limited to, the introduction of the polypeptide or variantsand fragments thereof directly into the plant or the introduction of atranscript into the plant. Such methods include, for example,microinjection or particle bombardment. See, e.g., Crossway et al.(1986) Mol Gen. Genet. 202: 179-185; Nomura et al. (1986) Plant Sci. 44:53-58; Hepler et al. (1994) Proc. Natl. Acad. Sci. 91: 2176-2180 andHush et al. (1994) The Journal of Cell Science 107: 775-784, all ofwhich are herein incorporated by reference. Alternatively, aherbicide-tolerance polynucleotide can be transiently transformed intothe plant using techniques known in the art. Such techniques includeviral vector system and the precipitation of the polynucleotide in amanner that precludes subsequent release of the DNA. Thus, thetranscription from the particle-bound DNA can occur, but the frequencywith which it is released to become integrated into the genome isgreatly reduced. Such methods include the use particles coated withpolyethylimine (PEI; Sigma #P3143).

In other embodiments, polynucleotides may be introduced into plants bycontacting plants with a virus or viral nucleic acids. Generally, suchmethods involve incorporating a nucleotide construct within a viral DNAor RNA molecule. It is recognized that a polypeptide of interest may beinitially synthesized as part of a viral polyprotein, which later may beprocessed by proteolysis in vivo or in vitro to produce the desiredrecombinant protein. Further, it is recognized that useful promoters mayinclude promoters utilized for transcription by viral RNA polymerases.Methods for introducing polynucleotides into plants and expressing apolypeptide encoded thereby, involving viral DNA or RNA molecules, areknown in the art. See, e.g., U.S. Pat. Nos. 5,889,191, 5,889,190,5,866,785, 5,589,367, 5,316,931, and Porta et al. (1996) MolecularBiotechnology 5: 209-221; herein incorporated by reference.

Methods are known in the art for the targeted insertion of apolynucleotide at a specific location in the plant genome. In oneembodiment, the insertion of the polynucleotide at a desired genomiclocation is achieved using a site-specific recombination system. See,e.g., WO99/25821, WO99/25854, WO99/25840, WO99/25855, and WO99/25853,all of which are herein incorporated by reference. Briefly, apolynucleotide can be contained in transfer cassette flanked by twonon-recombinogenic recombination sites. The transfer cassette isintroduced into a plant having stably incorporated into its genome atarget site which is flanked by two non-recombinogenic recombinationsites that correspond to the sites of the transfer cassette. Anappropriate recombinase is provided and the transfer cassette isintegrated at the target site. The polynucleotide of interest is therebyintegrated at a specific chromosomal position in the plant genome.

The cells that have been transformed may be grown into plants inaccordance with conventional ways. See, e.g., McCormick et al. (1986)Plant Cell Reports 5: 81-84. These plants may then be grown, and eitherpollinated with the same transformed strain or different strains, andthe resulting progeny having constitutive expression of the desiredphenotypic characteristic identified. Two or more generations may begrown to ensure that expression of the desired phenotypic characteristicis stably maintained and inherited and then seeds harvested to ensureexpression of the desired phenotypic characteristic has been achieved.In this manner, transformed seed (also referred to as “transgenic seed”)having a polynucleotide conferring tolerance to a PPO inhibitor stablyincorporated into their genome are provided.

In specific embodiments, a polypeptide or the polynucleotide of interestis introduced into the plant cell. Subsequently, a plant cell having theintroduced sequence is selected using methods known to those of skill inthe art such as, but not limited to, Southern blot analysis, DNAsequencing, PCR analysis, or phenotypic analysis. A plant or plant partaltered or modified by the foregoing embodiments is grown under plantforming conditions for a time sufficient to modulate the concentrationand/or activity of polypeptides in the plant. Plant forming conditionsare well known in the art and discussed briefly elsewhere herein.

It is also recognized that the level and/or activity of a polypeptide ofinterest may be modulated by employing a polynucleotide that is notcapable of directing, in a transformed plant, the expression of aprotein or an RNA. For example, the polynucleotides may be used todesign polynucleotide constructs that can be employed in methods foraltering or mutating a genomic nucleotide sequence in an organism. Suchpolynucleotide constructs include, but are not limited to, RNA:DNAvectors, RNA:DNA mutational vectors, RNA:DNA repair vectors,mixed-duplex oligonucleotides, self-complementary RNA:DNAoligonucleotides, and recombinogenic oligonucleobases. Such nucleotideconstructs and methods of use are known in the art. See, U.S. Pat. Nos.5,565,350; 5,731,181; 5,756,325; 5,760,012; 5,795,972; and 5,871,984;all of which are herein incorporated by reference. See also, WO98/49350, WO 99/07865, WO 99/25821, and Beetham et al. (1999) Proc.Natl. Acad. Sci. USA 96: 8774-8778; herein incorporated by reference.

It is therefore recognized that methods disclosed do not depend on theincorporation of the entire polynucleotide into the genome, only thatthe plant or cell thereof is altered as a result of the introduction ofthe polynucleotide into a cell. In one embodiment, the genome may bealtered following the introduction of the polynucleotide into a cell.For example, the polynucleotide, or any part thereof, may incorporateinto the genome of the plant. Alterations to the genome include, but arenot limited to, additions, deletions, and substitutions of nucleotidesinto the genome. While the methods disclosed do not depend on additions,deletions, and substitutions of any particular number of nucleotides, itis recognized that such additions, deletions, or substitutions comprisesat least one nucleotide.

Plants may be produced by any suitable method, including breeding. Plantbreeding can be used to introduce desired characteristics (e.g., astably incorporated transgene or a genetic variant or genetic alterationof interest) into a particular plant line of interest, and can beperformed in any of several different ways. Pedigree breeding startswith the crossing of two genotypes, such as an elite line of interestand one other elite inbred line having one or more desirablecharacteristics (i.e., having stably incorporated a polynucleotide ofinterest, having a modulated activity and/or level of the polypeptide ofinterest, etc.) which complements the elite plant line of interest. Ifthe two original parents do not provide all the desired characteristics,other sources can be included in the breeding population. In thepedigree method, superior plants are selfed and selected in successivefilial generations. After a sufficient amount of inbreeding, successivefilial generations will serve to increase seed of the developed inbred.In specific embodiments, the inbred line comprises homozygous alleles atabout 95% or more of its loci. Various techniques known in the art canbe used to facilitate and accelerate the breeding (e.g., backcrossing)process, including, for example, the use of a greenhouse or growthchamber with accelerated day/night cycles, the analysis of molecularmarkers to identify desirable progeny, and the like.

In addition to being used to create a backcross conversion, backcrossingcan also be used in combination with pedigree breeding to modify anelite line of interest and a hybrid that is made using the modifiedelite line. As discussed previously, backcrossing can be used totransfer one or more specifically desirable traits from one line, thedonor parent, to an inbred called the recurrent parent, which hasoverall good agronomic characteristics yet lacks that desirable trait ortraits. However, the same procedure can be used to move the progenytoward the genotype of the recurrent parent but at the same time retainmany components of the non-recurrent parent by stopping the backcrossingat an early stage and proceeding with selfing and selection. Forexample, an F1, such as a commercial hybrid, is created. This commercialhybrid may be backcrossed to one of its parent lines to create a BC1 orBC2. Progeny are selfed and selected so that the newly developed inbredhas many of the attributes of the recurrent parent and yet several ofthe desired attributes of the non-recurrent parent. This approachleverages the value and strengths of the recurrent parent for use in newhybrids and breeding.

Therefore, a method of making a backcross conversion of an inbred lineof interest comprising the steps of crossing a plant from the inbredline of interest with a donor plant comprising at least one mutant geneor transgene conferring a desired trait (e.g., herbicide tolerance),selecting an F1 progeny plant comprising the mutant gene or transgeneconferring the desired trait, and backcrossing the selected F1 progenyplant to a plant of the inbred line of interest is provided. This methodmay further comprise the step of obtaining a molecular marker profile ofthe inbred line of interest and using the molecular marker profile toselect for a progeny plant with the desired trait and the molecularmarker profile of the inbred line of interest. In the same manner, thismethod may be used to produce an F1 hybrid seed by adding a final stepof crossing the desired trait conversion of the inbred line of interestwith a different plant to make F1 hybrid seed comprising a mutant geneor transgene conferring the desired trait.

Recurrent selection is a method used in a plant breeding program toimprove a population of plants. The method entails individual plantscross pollinating with each other to form progeny. The progeny are grownand the superior progeny selected by any number of selection methods,which include individual plant, half-sib progeny, full-sib progeny,selfed progeny and topcrossing. The selected progeny arecross-pollinated with each other to form progeny for another population.This population is planted and again superior plants are selected tocross pollinate with each other. Recurrent selection is a cyclicalprocess and therefore can be repeated as many times as desired. Theobjective of recurrent selection is to improve the traits of apopulation. The improved population can then be used as a source ofbreeding material to obtain inbred lines to be used in hybrids or usedas parents for a synthetic cultivar. A synthetic cultivar is theresultant progeny formed by the intercrossing of several selectedinbreds.

Mass selection is a useful technique when used in conjunction withmolecular marker enhanced selection. In mass selection seeds fromindividuals are selected based on phenotype and/or genotype. Theseselected seeds are then bulked and used to grow the next generation.Bulk selection requires growing a population of plants in a bulk plot,allowing the plants to self-pollinate, harvesting the seed in bulk andthen using a sample of the seed harvested in bulk to plant the nextgeneration. Instead of self pollination, directed pollination could beused as part of the breeding program.

Mutation breeding is one of many methods that could be used to introducenew traits into an elite line. Mutations that occur spontaneously or areartificially induced can be useful sources of variability for a plantbreeder. The goal of artificial mutagenesis is to increase the rate ofmutation for a desired characteristic. Mutation rates can be increasedby many different means including temperature, long-term seed storage,tissue culture conditions, radiation such as X-rays, Gamma rays (e.g.,cobalt 60 or cesium 137), neutrons, (product of nuclear fission ofuranium 235 in an atomic reactor), Beta radiation (emitted fromradioisotopes such as phosphorus 32 or carbon 14), or ultravioletradiation (typically from 2500 to 2900 nm), or chemical mutagens (suchas base analogues (5-bromo-uracil), related compounds (8-ethoxycaffeine), antibiotics (streptonigrin), alkylating agents (sulfurmustards, nitrogen mustards, epoxides, ethyleneamines, sulfates,sulfonates, sulfones, lactones), azide, hydroxylamine, nitrous acid, oracridines. Once a desired trait is observed through mutagenesis thetrait may then be incorporated into existing germplasm by traditionalbreeding techniques, such as backcrossing. Details of mutation breedingcan be found in “Principals of Cultivar Development” Fehr, 1993Macmillan Publishing Company the disclosure of which is incorporatedherein by reference. In addition, mutations created in other lines maybe used to produce a backcross conversion of elite lines that comprisessuch mutations.

Methods of Modulating Expression

In some embodiments, the activity and/or level of the polypeptide ismodulated (i.e., increased or decreased). An increase in the leveland/or activity of the polypeptide can be achieved by providing thepolypeptide to the plant. As discussed elsewhere herein, many methodsare known the art for providing a polypeptide to a plant including, butnot limited to, direct introduction of the polypeptide into the plant,introducing into the plant (transiently or stably) a polynucleotideconstruct encoding a polypeptide having the desired activity.

Methods of Controlling Weeds

Methods are provided for controlling weeds in an area of cultivation,preventing the development or the appearance of herbicide resistantweeds in an area of cultivation, producing a crop, and increasing cropsafety. The term “controlling,” and derivations thereof, for example, asin “controlling weeds” refers to one or more of inhibiting the growth,germination, reproduction, and/or proliferation of and/or killing,removing, destroying, or otherwise diminishing the occurrence and/oractivity of a weed.

The PPO inhibitor plants display tolerance to herbicides and thereforeallow for the application of one or more herbicides at rates that wouldsignificantly damage control plants and further allow for theapplication of combinations of herbicides at lower concentrations thannormally applied which still continue to selectively control weeds. Inaddition, the PPO inhibitor-tolerant plants can be used in combinationwith herbicide blends technology and thereby make the application ofchemical pesticides more convenient, economical, and effective for theproducer.

The methods comprise planting the area of cultivation with PPOinhibitor-tolerant crop seeds or plants, and applying to any crop, croppart, weed or area of cultivation thereof an effective amount of a PPOinhibitor containing herbicide of interest. It is recognized that theherbicide can be applied before and/or after the crop is planted in thearea of cultivation. Such herbicide applications can include anapplication of any PPO inhibitor chemistry, or any combination thereof.In some examples, a diphenyl ether, triazolinone, N-phenylphthalimide,pyrimidindione and/or oxadiazole containing herbicide formulation isapplied. In some examples the herbicide formulation comprisesacifluorfen, fomesafen, oxyfluorfen, lactofen, sulfentrazone,carfentrasone, azafeniden flumiclorac, flumioxazin, oxadiazon,fluthiacet, benzfendizone, butagenacil and/or saflufenacil.

In other examples, the combination of herbicides comprises a glyphosate,a glufosinate, a dicamba, a bialaphos, a phosphinothricin, a protoxinhibitor, a sulfonylurea, an imidazolinone, a chlorsulfuron, animazapyr, a chlorimuron-ethyl, a quizalofop, an HPPD, a PPO inhibitor,and/or a fomesafen, or combinations thereof, wherein said effectiveamount is tolerated by the crop and controls weeds. Any effective amountof these herbicides can be applied, wherein the effective amount is anyamount that differentiates between plant cells, plants, and/or seedcomprising a PPO inhibitor tolerance allele, a PPO inhibitorpolynucleotide, and/or a polynucleotide encoding an ABC transporterprotein that confers tolerance to herbicide formulations comprising aPPO inhibitor. In some examples the herbicides are appliedsimultaneously, in some examples the herbicides are appliedsequentially, in some examples the herbicides are applied aspre-emergent treatments, in some examples the herbicides are applied aspost-emergent treatments, in some examples the herbicides are applied asa combination of pre- and post-emergent treatments.

In some examples, the method of controlling weeds comprises planting thearea with PPO inhibitor tolerant crop seeds or plants and applying tothe crop, crop part, seed of said crop or the area under cultivation, aneffective amount of a herbicide, wherein said effective amount comprisesan amount that is not tolerated by a control crop when applied to thecontrol crop, crop part, seed or the area of cultivation, wherein thecontrol crop does not express a polynucleotide that encodes anherbicide-tolerance polypeptide. In specific embodiments, combinationsof herbicides may be used, such as when an additional tolerance trait isincorporated into the plant.

In another embodiment, the method of controlling weeds comprisesplanting the area with a PPO inhibitor-tolerant crop seeds or plant andapplying to the crop, crop part, seed of said crop or the area undercultivation, an effective amount of a herbicide, wherein said effectiveamount comprises a level that is above the recommended label use ratefor the crop, wherein said effective amount is tolerated when applied tothe PPO inhibitor-tolerant crop, crop part, seed, or the area ofcultivation thereof.

Any herbicide can be applied to the tolerant crop, crop part, or thearea of cultivation containing said crop plant. Classifications ofherbicides (i.e., the grouping of herbicides into classes andsubclasses) is well-known in the art and includes classifications byHRAC (Herbicide Resistance Action Committee) and WSSA (the Weed ScienceSociety of America) (see also, Retzinger and Mallory-Smith (1997) WeedTechnology 11: 384-393). An abbreviated version of the HRACclassification (with notes regarding the corresponding WSSA group) isset forth below:

HRAC WSSA Group Mode of Action Chemical Family Active Ingredient Group AInhibition of acetyl Aryloxyphenoxy- clodinafop- 1 CoA carboxylasepropionate “FOPs” propargyl (ACCase) cyhalofop-butyl diclofop-methylfenoxaprop-P-ethyl fluazifop-P-butyl haloxyfop-R- methyl propaquizafopquizalofop-P-ethyl Cyclohexanedione alloxydim “DIMs” butroxydimclethodim cycloxydim profoxydim sethoxydim tepraloxydin tralkoxydimPhenylpyrazoline “DEN” pinoxaden B Inhibition of Sulfonylureaamidosulfuron 2 acetolactate azimsulfuron synthase ALSbensulfuron-methyl (acetohydroxyacid chlorimuron-ethyl synthase AHAS)chlorsulfuron cinosulfuron cyclosulfamuron ethametsulfuron- methylethoxysulfuron flazasulfuron flupyrsulfuron- methyl-Na foramsulfuronhalosulfuron- methyl imazosulfuron iodosulfuron mesosulfuronmetsulfuron-methyl nicosulforon oxasulforon primisulfuron- methylprosulfuron pyrazosulfuron- ethyl rimsulfuron sulfometuron- methylsulfosulfuron thifensulfuron- methyl triasulfuron tribenuron-methyltrifloxysulfuron triflusulfuron- methyl tritosulfuron Imidazolinoneimazapic imazamethabenz- methyl imazamox imazapyr imazaquin imazethapyrTriazolopyrimidine cloransulam-methyl diclosulam florasulam flumetsulammetosulam penoxsulam Pyrimidinyl(thio)benzoate bispyribac-Napyribenzoxim pyriftalid pyrithiobac-Na pyriminobac- methylSulfonylaminocarbonyl- flucarbazone-Na triazolinone propoxycarbazone- NaC1 Inhibition of Triazine ametryne 5 photosynthesis at atrazinephotosystem II cyanazine desmetryne dimethametryne prometon prometrynepropazine simazine simetryne terbumeton terbuthylazine terbutrynetrietazine Triazinone hexazinone metamitron metribuzin Triazolinoneamicarbazone Uracil bromacil lenacil terbacil Pyridazinone pyrazon =chloridazon Phenyl-carbamate desmedipham phenmedipham C2 Inhibition ofUrea chlorobromuron 7 photosynthesis at chlorotoluron photosystem IIchloroxuron dimefuron diuron ethidimuron fenuron fluometuron (see F3)isoproturon isouron linuron methabenzthiazuron metobromuron metoxuronmonolinuron neburon siduron tebuthiuron Amide propanil pentanochlor C3Inhibition of Nitrile bromofenoxim 6 photosynthesis at bromoxynilphotosystem II ioxynil Benzothiadiazinone bentazon Phenyl-pyridazinepyridate pyridafol D Photosystem-I- Bipyridylium diquat 22 electrondiversion paraquat E Inhibition of Diphenylether acifluorfen-Na 14protoporphyrinogen bifenox oxidase (PPO) chlomethoxyfen fluoroglycofen-ethyl fomesafen halosafen lactofen oxyfluorfen Phenylpyrazole fluazolatepyraflufen-ethyl N-phenylphthalimide cinidon-ethyl flumioxazinflumiclorac-pentyl Thiadiazole fluthiacet-methyl thidiazimin Oxadiazoleoxadiazon oxadiargyl Triazolinone azafenidin carfentrazone-ethylsulfentrazone Oxazolidinedione pentoxazone Pyrimidindione benzfendizonebutafenacil Other pyraclonil profluazol flufenpyr-ethyl F1 Bleaching:Pyridazinone norflurazon 12 Inhibition of carotenoid biosynthesis at thephytoene desaturase step (PDS) Pyridinecarboxamide diflufenicanpicolinafen Other beflubutamid fluridone flurochloridone flurtamone F2Bleaching: Triketone mesotrione 27 Inhibition of 4- sulcotrionehydroxyphenyl- pyruvate- dioxygenase (4- HPPD) Isoxazole isoxachlortoleisoxaflutole Pyrazole benzofenap pyrazolynate pyrazoxyfen Otherbenzobicyclon F3 Bleaching: Triazole amitrole 11 Inhibition of (in vivoinhibition carotenoid of lycopene cyclase biosynthesis (unknown target)Isoxazolidinone clomazone 13 Urea fluometuron (see C2) Diphenyletheraclonifen G Inhibition of EPSP Glycine glyphosate 9 synthase sulfosate HInhibition of Phosphinic acid glufosinate- 10 glutamine ammoniumsynthetase bialaphos = bilanaphos I Inhibition of DHP Carbamate asulam18 (dihydropteroate) synthase K1 Microtubule Dinitroaniline benefin = 3assembly inhibition benfluralin butralin dinitramine ethalfluralinoryzalin pendimethalin trifluralin Phosphoroamidate amiprophos-methylbutamiphos Pyridine dithiopyr thiazopyr Benzamide propyzamide =pronamide tebutam Benzoic acid DCPA = chlorthal- dimethyl K2 Inhibitionof Carbamate chlorpropham 23 mitosis/ propham microtubule carbetamideorganisation K3 Inhibition of Chloroacetamide acetochlor 15 VLCFAsalachlor (Inhibition of cell butachlor division) dimethachlordimethanamid metazachlor metolachlor pethoxamid pretilachlor propachlorpropisochlor thenylchlor Acetamide diphenamid napropamide naproanilideOxyacetamide flufenacet mefenacet Tetrazolinone fentrazamide Otheranilofos cafenstrole piperophos L Inhibition of cell Nitrile dichlobenil20 wall (cellulose) chlorthiamid synthesis Benzamide isoxaben 21Triazolocarboxamide flupoxam Quinoline carboxylic acid quinclorac (for26 monocots) (also group O) M Uncoupling Dinitrophenol DNOC 24 (Membranedinoseb disruption) dinoterb N Inhibition of lipid Thiocarbamatebutylate 8 synthesis - not cycloate ACCase inhibition dimepiperate EPTCesprocarb molinate orbencarb pebulate prosulfocarb thiobencarb =benthiocarb tiocarbazil triallate vernolate Phosphorodithioate bensulideBenzofuran benfuresate ethofumesate Chloro-Carbonic-acid TCA 26 dalaponflupropanate O Action like indole Phenoxy-carboxylic-acid clomeprop 4acetic acid 2,4-D (synthetic auxins) 2,4-DB dichlorprop = 2,4- DP MCPAMCPB mecoprop = MCPP = CMPP Benzoic acid chloramben dicamba TBA Pyridinecarboxylic acid clopyralid fluroxypyr picloram triclopyr Quinolinecarboxylic acid quinclorac (also group L) quinmerac Otherbenazolin-ethyl P Inhibition of auxin Phthalamate naptalam 19 transportSemicarbazone diflufenzopyr-Na Z Unknown (actual Arylaminopropionic acidFlamprop-M- 25 mode of action methyl/-isopropyl unknown, but likely thatthey differ in mode of action between themselves and from other groups)Pyrazolium difenzoquat 26 Organoarsenical DSMA 17 MSMA Other bromobutide27 (chloro)-flurenol cinmethylin cumyluron dazomet dymron = daimuronmethyl-dimuron = methyl-dymron etobenzanid fosamine indanofan metamoxaziclomefone oleic acid pelargonic acid pyributicarb

Herbicides can be classified by their mode of action and/or site ofaction and can also be classified by the time at which they are applied(e.g., pre-emergent or post-emergent), by the method of application(e.g., foliar application or soil application), or by how they are takenup by or affect the plant. Mode of action generally refers to themetabolic or physiological process within the plant that the herbicideinhibits or otherwise impairs, whereas site of action generally refersto the physical location or biochemical site within the plant where theherbicide acts or directly interacts. Herbicides can be classified invarious ways, including by mode of action and/or site of action. Often,a herbicide-tolerance gene that confers tolerance to a particularherbicide or other chemical on a plant expressing it will also confertolerance to other herbicides or chemicals in the same class orsubclass, for example, a class or subclass set forth in the table above.Thus, in some examples, a transgenic plant is tolerant to more than oneherbicide or chemical in the same class or subclass, such as, forexample, an inhibitor of PPO, a sulfonylurea, or a synthetic auxin. Insome examples the plant is transgenic for one or more of the herbicidetolerance traits, non-transgenic for one of more of the tolerancetraits, or any combination thereof.

Typically, the plants provided can tolerate treatment with differenttypes of herbicides (i.e., herbicides having different modes of actionand/or different sites of action) as well as with higher amounts ofherbicides than previously known plants, thereby permitting improvedweed management strategies that are recommended in order to reduce theincidence and prevalence of herbicide-tolerant weeds. Specific herbicidecombinations can be employed to effectively control weeds.

A transgenic crop plant which can be selected for use in crop productionbased on the prevalence of herbicide-tolerant weed species in the areawhere the transgenic crop is to be grown is provided. Methods are knownin the art for assessing the herbicide tolerance of various weedspecies. Weed management techniques are also known in the art, such asfor example, crop rotation using a crop that is tolerant to a herbicideto which the local weed species are not tolerant. A number of entitiesmonitor and publicly report the incidence and characteristics ofherbicide-tolerant weeds, including the Herbicide Resistance ActionCommittee (HRAC), the Weed Science Society of America, and various stateagencies (see, e.g., herbicide tolerance scores for various broadleafweeds from the 2004 Illinois Agricultural Pest Management Handbook), andone of skill in the art would be able to use this information todetermine which crop and herbicide combinations should be used in aparticular location.

These entities also publish advice and guidelines for preventing thedevelopment and/or appearance of and controlling the spread of herbicidetolerant weeds (see, e.g., Owen and Hartzler (2004), 2005 HerbicideManual for Agricultural Professionals, Pub. WC 92 Revised (Iowa StateUniversity Extension, Iowa State University of Science and Technology,Ames, Iowa); Weed Control for Corn, Soybeans, and Sorghum, Chapter 2 of“2004 Illinois Agricultural Pest Management Handbook” (University ofIllinois Extension, University of Illinois at Urbana-Champaign, Ill.);Weed Control Guide for Field Crops, MSU Extension Bulletin E434(Michigan State University, East Lansing, Mich.)).

Also included are plant cells, plants, and/or seeds produced by any ofthe foregoing methods.

The present invention is illustrated by the following examples. Theforegoing and following description of the present invention and thevarious embodiments are not intended to be limiting of the invention butrather are illustrative thereof. Hence, it will be understood that theinvention is not limited to the specific details of these examples.

EXAMPLES Example 1 Identification of Sulfentrazone Tolerant andSensitive Soybean Lines-Herbicide Screening Bioassay and IntergroupAssociation Marker Based Diagnostic

Sulfentrazone is a PPO inhibitor and is the active ingredient inAuthority® herbicide. Authority® 75DF (FMC Corp., Philadelphia, Pa.,USA) is a 75% active ingredient formulation of sulfentrazone containingno other active ingredients.

Part 1: Herbicide Bioassay

One hundred sixteen (116) elite soybean lines were screened forsulfentrazone tolerance using the following protocol. Seed of soybeanvarieties with adequate seed quality, having greater than 85% warmgermination were used.

Design and Replication:

After planting, entries were set up in a randomized complete blockdesign, blocked by replication. Three replications per experiment wereused. One or more of well established check variety were included in theexperiment, such as available public sector check lines.

Non-tolerant check: Pioneer 9692, Asgrow A4715Tolerant check: Pioneer 9584, Syngenta S5960Growing conditions were as follows (greenhouse/growth chamber): 16 hrphotoperiod @ 85° F. (w/75° nighttime set back). Lighting is critical tothe success of the screening as stated below.

Method of Screening:

Four inch plastic pots were filled with a high quality universal pottingsoil. Entries were planted 1 inch deep at the rate of 5 seeds/pot. Abar-coded plastic stake was used to identify each entry. After plantingthe pots were allowed to sit in greenhouse overnight to acclimate tosoil and improve germination. The following day a sulfentrazoneherbicide solution was slowly poured over each pot and allowed to evenlysoak through entire soil profile. This ensured that each seed wasexposed to an equal amount of sulfentrazone. Pots were placed onaluminum trays and placed in a greenhouse or growth chamber under highintensity light conditions with photosynthetic photon flux density(PPFD) of at least 500 μmol/m/s. Proper lighting conditions werenecessary for this screening due to the nature of the PPO inhibitorused. Pots were lightly watered so that herbicide was not leached fromthe soil profile. After soybean emergence the pots were watered bykeeping aluminum trays filled with ¾″ of water under each pot.

Herbicide Solution:

A) Mix a stock solution of 0.926 g Authority® 75DF (FMC Corp.),thoroughly dissolved in 1000 ml of water.B) Mix 10 ml of STOCK SOLUTION in 1000 ml of water to create finalsolution.C) Pour 100 ml of FINAL SOLUTION over each pot.

Recording Data:

10-14 days after treatment, plants were ready to be scored. All scoreswere based on a comparison to the checks and evaluated as follows:

-   -   9=Equivalent or better when compared to the tolerant check    -   7=Very little damage or response noted.    -   5=Intermediate response or damage    -   3=Major damage, including stunting and foliar necrosis    -   1=Severe damage, including severe stunting and necrosis;        equivalent or worse when compared to the non-tolerant check

Of the 116 soybean lines screened, 102 showed at least some tolerance tosulfentrazone based herbicides and 11 showed high sensitivity. Areference relevant to this protocol would be: Dayan et al. (1997)‘Soybean (Glycine max) cultivar differences in response tosulfentrazone’ Weed Science 45:634-641.

Part 2: Intergroup Analysis

An “Intergroup Allele Frequency Distribution” analysis was conductedusing GeneFlow™ version 7.0 software. An intergroup allele frequencydistribution analysis provides a method for finding non-randomdistributions of alleles between two phenotypic groups.

During processing, a contingency table of allele frequencies wasconstructed and from this a G-statistic and probability were calculated.The G statistic was adjusted by using the William's correction factor.The probability value was adjusted to take into account the fact thatmultiple tests are being done (thus, there is some expected rate offalse positives). The adjusted probability is proportional to theprobability that the observed allele distribution differences betweenthe two classes would occur by chance alone. The lower that probabilityvalue, the greater the likelihood that the tolerance phenotype and themarker will co-segregate. A more complete discussion of the derivationof the probability values can be found in the GeneFlow™ version 7.0software documentation. See also Sokal and Rolf (1981), Biometry: ThePrinciples and Practices of Statistics in Biological Research, 2nd ed.,San Francisco, W. H. Freeman and Co.

The underlying logic is that markers with significantly different alleledistributions between the tolerant and non-tolerant groups (i.e.,non-random distributions) might be associated with the trait and can beused to separate them for purposes of marker assisted selection ofsoybean lines with previously uncharacterized tolerance or non-toleranceto protoporphyrinogen oxidase inhibitors. The present analysis examinedone marker locus at a time and determined if the allele distributionwithin the tolerant group is significantly different from the alleledistribution within the non-tolerant group. A statistically differentallele distribution is an indication that the marker is linked to alocus that is associated with tolerance or non-tolerance toprotoporphyrinogen oxidase inhibitors. In this analysis, unadjustedprobabilities less than one are considered significant (the marker andthe phenotype show linkage disequilibrium), and adjusted probabilitiesless than approximately 0.05 are considered highly significant. Alleleclasses represented by less than 5 observations across both groups werenot included in the statistical analysis. In this analysis, 1043 markerloci had enough observations for analysis.

This analysis compares the plants' phenotypic score with the genotypesat the various loci. This type of intergroup analysis neither generatesnor requires any map data. Subsequently, map data (for example, acomposite soybean genetic map) is relevant in that multiple significantmarkers that are also genetically linked can be considered ascollaborating evidence that a given chromosomal region is associatedwith the trait of interest.

Results

Table 1 below provides a table listing the soybean markers thatdemonstrated linkage disequilibrium with the tolerance toprotoporphyrinogen oxidase inhibitor phenotype. There were 1043 markersused in this analysis. Also indicated in that table are the chromosomeson which the markers are located and their approximate map positionrelative to other known markers, given in cM, with position zero beingthe first (most distal) marker known at the beginning of the chromosome.These map positions are not absolute, and represent an estimate of mapposition. The statistical probabilities that the marker allele andtolerance phenotype are segregating independently are reflected in theAdjusted Probability values. Out of 584 loci studied in 38 sensitive and160 tolerant soybean lines, QTLs on Lg L and on Lg N were highlysignificant, as shown in the table below.

TABLE 1 Intergroup analysis results for LgL and LgN markers G- LocusTest Chrom# Position value df Prob(G) Adj Prob S00224-1 GW L 12.03 89.87−1 0 0 P10649C- ASH L 3.6 86.01 −1 0 0 3 SATT523 SSR L 32.4 24.02 −10.000001 0.000592 S60167- SSR N 26 62.35 −1 0 0 TB P5467A-1 ASH N 2516.25 −1 0.000056 0.032192 P5467A-2 ASH N 25 16.2 −1 0.000057 0.032731Table 2 below shows the allele distribution between 101 tolerant linesand 32/33 non-tolerant lines analyzed. Lines exhibiting tolerance areindicated in the first column as “TOL,” and lines exhibitingnon-tolerance are indicated in the first column as “NON.” Marker callsfor the P10649C-3 locus and the S60167-TB locus were available for 132and 63 of the lines respectively.

TABLE 2 Allele distribution P10649C-3 allele S60167-TB allele PhenotypeLG-L LG-N TOL 1 1 TOL 1 1 TOL 1 TOL 1 TOL 1 TOL 1 2 TOL 1 TOL 1 TOL 1 1TOL 1 TOL 1 1 TOL 1 TOL 1 1 TOL 1 1 TOL 1 1 TOL 1 1 TOL 1 TOL 1 1 TOL 1TOL 1 TOL 1 1 TOL 1 1 TOL 1 TOL 1 1 TOL 1 1 TOL 1 1 TOL 1 1 TOL 1 TOL 11 TOL 1 1 TOL 1 TOL 1 TOL 1 1 TOL 1 1 TOL 1 TOL 1 1 TOL 1 TOL 1 TOL 1TOL 1 1 TOL 1 1 TOL 1 1 TOL 1 1 TOL 1 TOL 1 1 TOL 1 1 TOL 1 TOL 1 1 TOL1 TOL 1 TOL 1 1 TOL 1 1 TOL 1 TOL 1 1 TOL 1 1 TOL 1 1 TOL 1 1 TOL 1 TOL1 1 TOL 1 1 TOL 1 TOL 1 TOL 1 TOL 1 TOL 1 TOL 1 TOL 1 1 TOL 1 TOL 1 1TOL 1 1 TOL 1 TOL 1 TOL 1 TOL 1 TOL 1 1 TOL 1 TOL 1 TOL 1 TOL 1 TOL 1TOL 1 1 TOL 1 1 TOL 1 1 TOL 1 TOL 1 TOL 1 1 TOL 1 TOL 1 TOL 1 TOL 1 TOL1 TOL 1 TOL 1 TOL 1 TOL TOL 1 TOL 1 TOL 2 2 TOL 1 TOL 1 TOL 1 NON 3 2NON 3 NON 1 1 NON 1_2 2 NON 3 2 NON 3 NON 1 1_2 NON 3 2 NON 2 1_2 NON 32 NON 2 NON 2 2 NON 2 2 NON 1 1 NON 2_3 NON 3 2 NON 3 2 NON 2_3 NON 3NON 3 NON 1 1 NON 2 NON 3 NON 3 2 NON 3 NON 2 2 NON 3 2 NON 1_3 2 NON 3NON 2 NON 1 NON 3

The non-random distribution of alleles between the tolerant andnon-tolerant plant groups at the marker loci in Table 2 is good evidencethat a QTL influencing tolerance to protoporphyrinogen oxidaseinhibitors is linked to these marker loci.

Example 2 Predication and Confirmation of Marker Based Selection forResponse to PPO Chemistries in a Set of Diverse Public Soybean Lines

Marker haplotype data for a set of 17 diverse public soybean lines wasdetermined for two QTL identified in Example 1 for Linkage Group Lmolecular markers P10649C-3 (approximate position 3.6) and S00224-1(approximate position 12.0). The response of these lines tosulfentrazone herbicide was published by Hulting et al. (Soybean(Glycine max (L.) Merr.) cultivar tolerance to sulfentrazone. 2001Science Direct, Vol. 20(8): 679-683). The phenotypic response wasreported as a growth reduction index: plant height and visual injury asexpressed as a percentage of check plot of each cultivar. Data for themarker haplotype on Linkage Group L and the herbicide bioassay resultsare presented in Table 3. Use of the molecular diagnostic P10649C-3(linked QTL on Linkage Group L, approximate position 3.6) for this setof phentoyped soybean lines is 92% predictive of tolerance tosulfentrazone when injury is set at 39% or less GRI and is 100%predictive of non-tolerance to sulfentrazone when injury is set at 40%or higher GRI. Use of the S00224-1 marker (approximate position 12.0)for this set of soybean lines is 88% predictive of tolerance tosulfentrazone when injury is set at 39% or less GRI and is 100%predictive of non-tolerance to sulfentrazone when injury is set at 40%or more GRI.

TABLE 3 Marker haplotype at/near QTL on Linkage Group L for PPOherbicide (sulfentrazone) response and phenotypic measure of cropresponse, expressed in terms of Growth Reduction Index, for soybeancultivars (italicized items indicate deviations from expected) GrowthLinkage Group L QTLs Reduction Position 3.6 Position 12.0 CultivarIndex* P10649C-3 S00224-1 PI88788 2 1, 1 3, 3 Richland 4 1, 1 3, 3Lincoln 5 1, 1 3, 3 PI180501 8 1, 1 3, 3 Illini 8 1, 1 3, 3 S100 8 1, 13, 3 Mukden 8 1, 1 3, 3 Arksoy 10 1, 1 3, 3 Capital 10 1, 1 3, 3Haberlandt 10 3, 3 2, 2 Ralsoy 13 1, 1 2, 3 Dunfield 16 1, 1 3, 3 Peking22 1, 1 3, 3 Roanoke 40 3, 3 2, 2 Ogden 42 3, 3 2, 2 Hutcheson 46 3, 32, 2 Ransom 52 3, 3 2, 2 allele call load percent accuracy correct(alleles 1) (allele 3) tolerant 24/26 = 92% 23/26 = 88% correct (allele3) (allele 2) = non-tolerant 8/8 = 100% 8/8 = 100% *growth reductionindex (plant height and visual injury as expressed as a percentage ofcheck plot of each cultivar); Pre-emergence sulfentrazone application of0.28 kg ai/ha, from Hulting, et al. (supra)

Haplotype data for a set of 15 diverse public soybean lines wasdetermined for two QTL identified in Example 1 for Linkage Group Nmolecular marker S60167 (approximate position 26.0). The response ofthese 15 lines to sulfentrazone herbicide was determined and publishedupon by Hulling et al. (Soybean (Glycine max (L.) Merr.) cultivartolerance to sulfentrazone. 2001 Science Direct, Vol. 20(8): 679-683).The phenotypic response was reported as a growth reduction index: plantheight and visual injury as expressed as a percentage of check plot ofeach cultivar. Data for the marker haplotype on Linkage Group N and theherbicide bioassay results are presented in Table 4. The cultivar Ralsoyis heterozygous for the S60167 marker. Use of the S60167 marker for thisset of phentoyped soybean lines is 88% predictive of tolerance tosulfentrazone when injury is set at 39% or less GRI and is 100%predictive of tolerance to sulfentrazone when injury is set at 40% orhigher GRI.

TABLE 4 Marker haplotype at/near QTL on Linkage Group N for PPOherbicide (sulfentrazone) response and phenotypic measure of cropresponse, expressed in terms of Growth Reduction Index, for soybeancultivars (italicized items indicate deviations from expected) GrowthLinkage Group N QTL Reduction Position 26 Cultivar Index* S60167-TBPI88788 2 1, 1 Richland 4 1, 1 Lincoln 5 1, 1 Illini 8 1, 1 S100 8 1, 1Mukden 8 1, 1 Arksoy 10 1, 1 Haberlandt 10 1, 1 Ralsoy 13 1, 2 Dunfield16 1, 1 CNS 20 2, 2 Peking 22 1, 1 Roanoke 40 2, 2 Ogden 42 2, 2Hutcheson 46 2, 2 allele call load percent accuracy correct (allele 1)tolerant 21/24 = 88% correct (allele 2) non-tolerant 6/6 = 100%

Example 3 Predication and Confirmation of Marker Based Selection forResponse to PPO Chemistries in a Set of Soybean Commercial Lines

Haplotype data for a set of 7 commercial soybean lines was determinedfor two QTL identified in the previous example for Linkage Group Lmolecular markers P10649C-3 (position 3.6) and S00224-1 (position 12.0).The response of these lines to sulfentrazone herbicide was determined bymethod used in Example 1. In addition, the same scale was used forscoring such that:

-   -   9=Equivalent or better when compared to the tolerant check    -   7=Very little damage or response noted.    -   5=Intermediate response or damage    -   3=Major damage, including stunting and foliar necrosis    -   1=Severe damage, including severe stunting and necrosis;        equivalent or worse when compared to the non-tolerant check        Data for the marker haplotype on Linkage Group L and the        herbicide bioassay results are presented in Table 5. Use of        either/both of these markers for this set of phentoyped soybean        lines is 100% predictive of both tolerance (score of a 7 or 9)        and non-tolerance (score of a 1 for the non-tolerant check).

TABLE 5 Prediction and confirmation of marker based selection at QTL forlinkage group L for response to PPO chemistry (sulfentrazone) in a setof commercial soybean varieties. sulfentrazone Position 3.6 Position12.0 Variety injury score P10649C-3 S00224-1 93B41 9 1, 1 3, 3 93B82 91, 1 3, 3 9281 9 1, 1 3, 3 9584 9 1, 1 3, 3 92B52 7 1, 1 3, 3 92B61 7 1,1 3, 3 9692 1 3, 3 2, 2

Example 4 Predication and Confirmation of Marker Based Selection forResponse to PPO Chemistries (Sulfentrazone) in Ten Lines from a Set ofSoybean Lines Phenotyped at the University of Illinois

A comparison for the marker predictiveness of PPO response wasconducted. The herbicide bioassay experiment used is described inPhytoxic Response and Yield of Soybean (Glycine max) Varieties Treatedwith Sulfentrazone or Flumioxazin (Taylor-Lovell et al., 2001 WeedTechnology 15:96-102). Phenotypic data was taken from Table 2 of thepublication for those varieties for which in-house marker data wasavailable. Phenotypic score and haplotype data for a set of 10 soybeanlines (1 public and 9 commercial) in the chromosomal regions around theQTL for Linkage group L is presented in Table 6. The phenotypic scorewas determined as percent injury which is defined as visible injuryratings including stunting, chlorosis, and bronzing symptomology (0=noinjury; 100=complete death) with 448 g ai/ha field application. Ratingswere taken 12 days after treatment. Use of marker P10649C (linked QTL onLinkage Group L, approximate position 3.6, allele call 1) for this setof phentoyped soybean lines is 100% predictive of tolerance (allelecall 1) to sulfentrazone when injury is 21% or less and is 100%predictive of non-tolerance (allele call 2 or 3) to sulfentrazone wheninjury is 43% or greater. The predictiveness of marker S00224-1 is also100% accurate for tolerance (allele 3) and non-tolerance (allele 2) forthis set of material.

TABLE 6 Marker haplotype at/near QTL on Linkage Group L for PPOherbicide (sulfentrazone) response and phenotypic measure of crop injurysulfentrazone Position 3.6 Position 12.0 Variety injury score P10649C-3S00224-1 P9584 5 1, 1 3, 3 P9671 5 1, 1 3, 3 P9151 8 1, 1 3, 3 P9306 151, 1 3, 3 Elgin 18 1, 1 3, 3 P9282 19 1, 1 3, 3 P9352 21 1, 1 3, 3 P936243 2, 2 2, 2 91B01 58 3, 3 2, 2 P9552 61 3, 3 2, 2 LSD (0.05) 8 allelecall load percent accuracy correct tolerant (alleles 1 or 2) (allele 3)14/14 = 100% 14/14 = 100% correct non-tolerant (allele 3) 8/8 = (allele2) = 100% 8/8 = 100%

Example 5 Pictures of Soybean Variety Response (Tolerant andNon-Tolerant Check Varieties) to Sulfentrazone Injury in the Field andin the Greenhouse/Growth Chamber Bioassay

Known non-tolerant (i.e., Pioneer variety 9692, Asgrow variety A4715)and tolerant (i.e., Pioneer variety 9584, Syngenta variety S5960)germplasm can exhibit severe differences in symptomology when fieldconditions are conducive to damage and when lab conditions for bioassaysare optimized for selection purposes. FIGS. 5 and 6 show thesedifferences in phenotype. FIG. 5 shows a field sample, with anon-tolerant variety on the left (stunted, necrotic) and tolerantvariety on the right (normal growth). FIG. 6 shows a greenhouse sample,with non-tolerant (left side) and tolerant (right side) variety checks,treated in the foreground, untreated in the background.

Example 6 Fine Mapping of PPO Herbicide Tolerance QTL

The PPO herbicide tolerance QTL was mapped in two mapping populations,GEID1653063×GEID3495695 and GEID4520632×GEID7589905 PPO tolerance mappedto chromosome GM19 (LG-L) near the closely linked marker S03859-1-A,which explains 80% of the phenotypic variation. From these twopopulations, lines with recombination breakpoints near S03859-1-A wereidentified to define the borders of the QTL and to facilitatefine-mapping.

Subsequent analysis of the recombinants indicated that the closelylinked marker S03859-1-A was actually the left flanking marker. TheGEID1653063×GEID3495695 population had 37 recombinants that set theflanking markers for the PPO QTL as 504867-1-A (GM19: 841543-841958) andS03859-1-A (GM19: 1634882-1635399) (Table 10). TheGEID4520632×GEID7589905 population had 42 recombinants that delimit theQTL to the same interval (Table 11).

Because S03859-1-A was determined to be closely linked to the PPO QTL,annotated loci in the vicinity of this marker were targeted for SNPdiscovery and marker development. Primers were designed from target lociusing Primer3 and checked for uniqueness using bioinformatics software.A panel composed of 20 PPO tolerant and 8 PPO susceptible lines,including the four mapping parents from the mapping population, wasre-sequenced at the target loci to identify informative SNPs. DNA wasextracted using the urea extraction protocol below and PCR amplifiedusing standard lab protocols (see Tables 7-8). The PCR was then cleanedup using the ExoSAP-IT® protocol (USB-Cleveland, Ohio, USA) (Table 9)before being sequenced by Sanger sequencing.

In total, 104 loci were re-sequenced and 235 informative SNPs wereidentified. From these SNPs, 22 Taqman® probe markers were designed todistinguish between tolerant versus susceptible alleles in the mappingpopulations. Taqman® assays were designed generally following ABIsuggested parameters. These markers were then run on the recombinantsfrom the two mapping populations to facilitate fine-mapping and tofurther delimit the PPO QTL interval.

Urea Extraction Protocol

-   -   1. Grind 2 g fresh tissue or 0.5 g lyophilized tissue and add it        to 6 mL Urea Extraction Buffer and mix well.    -   2. Add RNase A (100 mg/mL) and incubate @ 37° C. for 20 min.        -   a. 3 uL—Leaf        -   b. 12 uL—Seed    -   3. Add 4-5 mL Phenol:Chloroform:Isoamyl 25:24:1. Mix well.        (Sigma P3803)    -   4. Put on rocker inside hood.        -   a. Fresh—15 min        -   b. Lyophilized—30 min    -   5. Centrifuge @ 8000 rpm at 10° C. for 10 min.    -   6. Transfer supernatant to clean tube.    -   7. Add 700 uL of 3M NaOAC (pH 5.0) and 5 mL cold isopropanol.        Mix well.    -   8. Hook DNA and wash in 70% EtOH.    -   9. Repeat 70% wash.    -   10. Transfer pellet to 1.5 mL tube and allow to dry.    -   11. Dissolve pellet in 1 mL 10 mM Tris.

7 M Urea Extraction Buffer

Water 350 mL Urea 336 g 5 M NaCl 50 mL (14.61 g) 1 M Tris 40 mL (pH 8.0).5 M EDTA 32 mL (pH 8.0) 20% Sarcosine Sol. 40 mL (8 g) Adjust volume to800 mL with ddH2O

TABLE 7 PCR Reaction Mix for SNP Discovery. 1X 24 plate 36 plate 48plate (uL) (u1) (uL) (uL) gDNA (~50-100 ng) 2.0 — — — 10x PCR Buffer 2.05,952 7,680 10,944 1 mM dNTP 2.0 5,952 7,680 10,944 Taq 0.1 297.6 384547.2 0.5 uM Primer (F + R) 4.0 — — — ddH2O 9.9 29,462 38,016 54,173Total 20.0 41,664 53,760 76,608

TABLE 8 PCR Setup for SNP Discovery. Dipper Setup PCR conditions TempTime #Cycles initial denature 94 C. 3 min 1X denature 94 C. 45 secanneal 65 C. 60 sec 35X  extension 72 C. 75 sec final extension 72 C. 5min 1X end

TABLE 9 Exo/SAP Protocol for PCR clean up. PCR clean up Exo/SAP Mix(pre-sequencing) add 3.6 ul of mastermix to 7 μ1 final PCR product 24plate (μl) 36 plate (μl) 48 plate (μl) ddH2O 4,285.4 5,944.3 7,326.7 SAP4,285.4 5,944.3 7,326.7 Exo 2,142.7 2,972.2 3,663.4 total 10,714 14,86118,317

TABLE 10 Initial recombinants identified from GEID1653063 × GEID3495695mapping population that delimited PPO herbicide tolerance QTL tointerval between S01659-1-A and S03859-1-A. SAMPLE S04867-1-A S03859-1-AGenetic Pos 12.55 16.08 Call Average Comment GEID1653063 A A SUS 1Control GE1D3495695 B B TOL 9 Control SJ22185567 A B TOL 9 L borderSJ22185980 A B TOL 9 L border SJ22186045 A B TOL 9 L border SJ22186929 AB TOL 9 L border SJ22186019 B H TOL 9 R border SJ22185608 H B TOL 9 Lborder SJ22186913 H B TOL 9 L border SJ22185928 H B TOL 9 L borderSJ22186923 H B TOL 8.333333 L border SJ22185569 A H SEG 5 L borderSJ22186052 A H SEG 6.333333 L border SJ22186882 A H SEG 5 L borderSJ22186919 B H SEG 5.666667 L border SJ22186968 B H SEG 6.333333 Lborder SJ22186824 B H SEG 6.333333 L border SJ22185604 H B SEG 6.333333R border SJ22185573 H A SEG? 3.666667 R border SJ22185983 A B SUS 1 Rborder SJ22186894 A B SUS 2.333333 R border SJ22185562 A H SUS 1.666667R border SJ22185941 A H SUS 1 R border SJ22185534 B A SUS 3 L borderSJ22185545 B A SUS 1.666667 L border SJ22185559 B A SUS 2.333333 Lborder SJ22186023 B A SUS 3 L border SJ22186057 B A SUS 1 L borderSJ22186065 B A SUS 1 L border SJ22186837 B A SUS 3 L border SJ22185957 BA SUS 1 L border SJ22186846 B A SUS 1.666667 L border SJ22186840 H A SUS1 L border SJ22186950 H A SUS 1 L border SJ22186872 H A SUS 2.333333 Lborder SJ22186836 H A SUS 1.666667 L border SJ22186074 H A SUS 1 Lborder SJ22186906 H A SUS 1 L border SJ22185984 H A SUS 1 L border

TABLE 11 Initial recombinants identified from GEID4520632 × GEID7589905mapping population that delimited PPO herbicide tolerance QTL tointerval between S04867-1-A and S03859-1-A SAMPLE S04867-1-A S03859-1-AGenetic Pos 12.55 16.08 Call Ave Comment GEID7589905 A A SUS 1 ControlGEID4520632 B B TOL 9 Control SP21669231 A B TOL 9 L border SP21669401 AB TOL 9 L border SP21669240 A B TOL 9 L border SP21669613 A B TOL 9 Lborder SP21669249 H B TOL 9 L border SP21669645 H B TOL 9 L borderSP21669670 H B TOL 9 L border SP21669563 H B TOL 9 L border SP21669592 HB TOL 9 L border SP21669260 B A SUS 1 L border SP21669265 B A SUS 1 Lborder SP21669778 B A SUS 1.666667 L border SP21669590 B A SUS 1 Lborder SP21669751 A H SUS 1 R border SP21669380 H A SUS 2.666667 Lborder SP21669679 H A SUS 1 L border SP21669708 H A SUS 1 L borderSP21669755 H A SUS 1 L border SP21669214 H A SUS 1 L border SP21669573 HA SUS 1.666667 L border SP21669612 H A SUS 2.333333 L border SP21669336H A SUS 3.666667 L border SP21669201 B H SEG 5 L border SP21669503 B HSEG 5 L border SP21669664 B H SEG 5 L border SP21669540 B H SEG 5 Lborder SP21669752 B H SEG 5.666667 L border SP21669230 B H SEG 5.666667L border SP21669331 A H SEG 6.333333 L border SP21669371 A H SEG 5 Lborder SP21669542 A H SEG 6.333333 L border SP21669584 A H SEG 5 Lborder SP21669694 A H SEG 5.666667 L border SP21669763 A H SEG 5 Lborder SP21669533 A H SEG 5 L border SP21669417 A H SEG 6.333333 Lborder SP21669647 A H SEG? 7.666667 L border SP21669651 A H SEG?7.666667 L border SP21669541 H B SEG? 7.666667 R border SP21669749 H ASEG 5 R border SP21669356 H A SEG 5 R border SP21669674 H A SEG?3.666667 R border

From the GEID1653063×GEID3495695 mapping population, four keyrecombinants were identified which served to further fine-map the PPOQTL interval (Table 13). A recombination breakpoint at S08110-1-Q1 inline SJ22186052 set the left border, while breakpoints at S08105-1-Q1 inSJ22186019, SJ22186894, and SJ22185941 set the right border. Theserecombinants delimit the PPO QTL to ˜70 kb interval. From theGEID4520632×GEID7589905 mapping population, eight key recombinants wereidentified (Table 14). A recombination breakpoint in line SP21669503 atS08117-1-Q1 set the left border, while breakpoints in SP21669249,SP21669332, SP21669615, SP21669616, SP21669670, SP21669458, andSP21669760 set the right border at S08010-1-Q1. These recombinantsdelimit the PPO QTL to a ˜526 kb interval. However, when the data fromthese two mapping populations are combined into a single set, the PPOQTL interval is delimited to a ˜56 kb interval between S08117-1-Q1 andS08105-1-Q1 (Table 12).

TABLE 12 Summary of SNP markers used for initial QTL mapping andfine-mapping of PPO herbicide tolerance QTL. Combined data from the twopopulations delimits the QTL to a ~56 kb interval between S08117-1-Q1and S8105-1-Q1. First Base Last base Marker Amplicon Loci Coordinatecoordinate Population Fine-mapping Comment S04867-1-A Glyma19g01220.1841543 841958 Both S08102-1-Q1 PPO_Gm19_1487k3-1 Glyma19g01860.1 14891131489545 Both S08103-1-Q1 PPO_Gm19_1491k1-1 X 1491603 1492136 BothS08104-1-Q1 PPO_Gm19_1491k2-1 Glyma19g01870.1 1492364 1492948 BothS08106-1-Q1 PPO_Gm19_1499k2-1 Glyma19g01880.1 1500732 1501392GEID1653063/ GEID3495695 S08107-1-Q1 PPO_Gm19_1541k3-1 Glyma19g01900.11542880 1543693 GEID1653063/ GEID3495695 S08109-1-Q1 PPO_Gm19_1541k4-1Glyma19g01900.1 1543868 1544588 GEID1653063/ GEID3495695 S08110-1-Q1PPO_Gm19_1548k1-1 Glyma19g01910.1 1548367 1548822 GEID1653063/ L borderGEID3495695 GEID1653063/ GEID3495695 S08111-1-Q1 PPO_Gm19_1548k2-1Glyma19g01910.1 1548902 1549558 GEID1653063/ GEID3495695 S08115-2-Q1PPO_Gm19_1563k1-1 X 1563958 1564512 Both S08117-1-Q1 PPO_Gm19_1563k2-1 X1564563 1564960 Both L border GEID4520632/ GEID7589905 S08119-1-Q1PPO_Gm19_1566k2-1 Glyma19g01920.1 1567791 1568282 Both histonedeacetylase S08118-1-Q1 PPO_Gm19_1566k4-1 Glyma19g01920.1 15692731569748 Both histone deacetylase S08116-1-Q1 PPO_Gm19_1566k5-1Glyma19g01920.1 1570198 1570729 Both histone deacetylase S08101-1-Q1PPO_Gm19_1586k1-1 Glyma19g01940.1 1587051 1587687 Both multidrug/pheromone exporter, ABC superfamily S08112-1-Q1 PPO_Gm19_1586k1-1Glyma19g01940.1 1587051 1587687 Both multidrug/ pheromone exporter, ABCsuperfamily S08108-1-Q1 PPO_Gm19_1586k2-1 Glyma19g01940.1 15878051588500 Both multidrug/ pheromone exporter, ABC superfamily S08101-1-Q1PPO_Gm19_1586k4-1 Glyma19g01940.1 1589409 1590062 Both multidrug/pheromone exporter, ABC superfamily S08101-2-Q1 PPO_Gm19_1586k4-1Glyma19g01940.1 1589409 1590062 Both multidrug/ pheromone exporter, ABCsuperfamily S08101-3-Q1 PPO_Gm19_1586k4-1 Glyma19g01940.1 15894091590062 Both multidrug/ pheromone exporter, ABC superfamily S08101-4-Q1PPO_Gm19_1586k4-1 Glyma19g01940.1 1589409 1590062 Both multidrug/pheromone exporter, ABC superfamily S08105-1-Q1 PPO_Gm19_1618k2-1 X1619657 1620279 Both R border GEID1653063/ GEID3495695 S03859-1-Asbacm.pk005.c3.f X 1634882 1635399 Both S08010-1-Q1 PPO_Gm19_2089k4-1Glyma19g02370.1 2091644 2092359 Both R border GEID4520632/ GEID7589905S08010-2-Q2 PPO_Gm19_2089k4-1 Glyma19g02370.1 2091644 2092359 BothTables 13A-13G: Fine-mapping of the PPO herbicide tolerance QTL intervalwith recombinants from the GEID1653063×GEID3495695 population. Keyrecombinants delimit the QTL to the ˜70 kb interval between S08110-1-Q1and S08105-1-Q1.

TABLE 13A S08104- 1-Q1 Marker S08102-1-Q1 S08103-1-Q1 PPO_ Amplicon/PosS04867-1-A PPO_Gm19_ PPO_Gm19_ Gm19_ Sample Gm19: 841750 1487k3-11491k1-1 1491k2-1 SJ22185925 B B B B SJ22186974 B B B B SJ22185946 B B BB SJ22186019 B B B B SJ22186923 H H H H SJ22185604 H H H H SJ22186029 HH H H SJ22186052 A A A A SJ22185534 B A A A SJ22185552 A A A ASJ22186842 A A — A SJ22186924 A A A A SJ22186873 A A A A SJ22186894 A AA A SJ22185957 B A A A SJ22185941 A A A A SJ22186872 H A A A SJ22185984H H H H

TABLE 13B S08110- 1-Q1 Marker S08106-1-Q1 S08107-1-Q1 S08109-1-Q1 PPO_Amplicon/Pos PPO_Gm19_ PPO_Gm19_ PPO_Gm19_ Gm19_ Sample 1499k2-11541k3-1 1541k4-1 1548k1-1 SJ22185925 B2 B B B SJ22186974 B1 B B BSJ22185946 B2 B B B SJ22186019 B2 B B B SJ22186923 H B — B SJ22185604 HH H H SJ22186029 H H H H S322186052 A A A A SJ22185534 A A A ASJ22185552 A A A A SJ22186842 A A A A SJ22186924 A A A A SJ22186873 A AA A SJ22186894 H A A A SJ22185957 A A A A SJ22185941 A A A A SJ22186872A A A A SJ22185984 H A A A

TABLE 13C S08119- 1-Q1 Marker S08111-1-Q1 S08115-2-Q1 S08117-1-Q1 PPO_Amplicon/Pos PPO_Gm19_ PPO_Gm19_ PPO_Gm19_ Gm19_ Sample 1548k2-11563k1-1 1563k2-1 1566k2-1 SJ22185925 B B B B SJ22186974 B B/H B BSJ22185946 B B B B SJ22186019 B B B B SJ22186923 B B B B SJ22185604 H HH H SJ22186029 H H H H SJ22186052 — H H H SJ22185534 A A A A SJ22185552A A A A SJ22186842 A A A A SJ22186924 A A A A SJ22186873 A A A ASJ22186894 A A A A SJ22185957 A A A — SJ22185941 A A A A SJ22186872 A AA — SJ22185984 A A A A

TABLE 13D Marker S08118-1-Q1 S08116-1-Q1 S08101-1-Q1 S08112-1-Q1Amplicon/Pos PPO_Gm19_ PPO_Gm19_ PPO_Gm19_ PPO_Gm19_ Sample 1566k4-11566k5-1 1586k1-1 1586k1-1 SJ22185925 B B B B SJ22186974 — B B BSJ22185946 B B B B SJ22186019 — B B B SJ22186923 B B B B SJ22185604 H HH H SJ22186029 H H H H SJ22186052 — H H H SJ22185534 A A A A SJ22185552A A A A SJ22186842 A A A A SJ22186924 A A A A SJ22186873 A A A ASJ22186894 A A A A SJ22185957 A A A A SJ22185941 A A A A SJ22186872 A AA A SJ22185984 A A A A

TABLE 13E Marker S08108-1-Q1 S08101-1-Q1 S08101-2-Q1 S08101-3-Q1Amplicon/Pos PPO_Gm19_ PPO_Gm19_ PPO_Gm19_ PPO_Gm19_ Sample 1586k2-11586k4-1 1586k4-1 1586k4-1 SJ22185925 B B B B SJ22186974 B B B BSJ22185946 B B B B SJ22186019 B B B B SJ22186923 B B B B SJ22185604 H HH H S122186029 H H H H SJ22186052 H H H H SJ22185534 A A A A SJ22185552A A A A SJ22186842 A A A A SJ22186924 A A A A SJ22186873 A A A ASJ22186894 A A A A SJ22185957 A A A A SJ22185941 A A A A SJ22186872 A AA A SJ22185984 A A A A

TABLE 13F S08010-1-Q1 Marker S08101-4-Q1 S08105-1-Q1 S03859-1-A PPO_Amplicon/Pos PPO_Gm19_ PPO_Gm19_ PPO_Gm19_ Gm19_20 Sample 1586k4-11618k2-1 1635140 89k4-1 SJ22185925 B B B A SJ22186974 B B B A SJ22185946B B B A SJ22186019 B H H H SJ22186923 B B B B SJ22185604 H B B BSJ22186029 H H H B SJ22186052 H H H H SJ22185534 A A A B SJ22185552 A AA B SJ22186842 A A A B SJ22186924 A A A B SJ22186873 A A A B SJ22186894A B B B SJ22185957 A A A B SJ22185941 A H H H SJ22186872 A A A BSJ22185984 A A A A

TABLE 13G Marker S08010-2-Q2 Amplicon/ PPO_Gm19_ Pos 2089k4-1 CommentPhenotype SJ22185925 A TOL SJ22186974 A TOL SJ22185946 H TOL SJ22186019H R Border TOL SJ22186923 B TOL SJ22185604 B SEG SJ22186029 B SEGSJ22186052 H L Border SEG SJ22185534 B SUS SJ22185552 B SUS SJ22186842 BSUS SJ22186924 B SUS SJ22186873 B SUS SJ22186894 B R Border SUSSJ22185957 B SUS SJ22185941 H R Border SUS SJ22186872 B SUS SJ22185984 ASUSTables 14A-14C: Fine-mapping of the PPO herbicide tolerance QTL intervalwith recombinants from the GEID4520632×GEID7589905 population.

TABLE 14A Marker Sample Comment Phenotype S04867-1-A S08102-1-Q1S08103-1-Q1 S08104-1-Q1 S08115-2-Q1 S08117-1-Q1 SP21669249 R Border TOLH B B B B B SP21669332 R Border TOL B B — B B B SP21669615 R Border TOLB — B B B B SP21669616 R Border TOL B B — B B/H B SP21669670 R BorderTOL H B B B — B SP21669503 L Border SEG B B B B B B SP21669458 R BorderSUS A A A A A A SP21669760 R Border SUS A A A A A A

TABLE 14B Marker Sample Comment Phenotype S08119-1-Q1 S08118-1-Q1S08116-1-Q1 S08101-1-Q1 S08112-1-Q1 S08108-1-Q1 SP21669249 R Border TOLB B B B B B SP21669332 R Border TOL B B B B B B SP21669615 R Border TOLB B B B B B SP21669616 R Border TOL B B B B B/H B SP21669670 R BorderTOL B B B B B B SP21669503 L Border SEG H H H H H H SP21669458 R BorderSUS A A A A A A SP21669760 R Border SUS A A A A A A

TABLE 14C Marker Sample Comment Phenotype S08101-1-Q1 S08101-2-Q1S08101-3-Q1 S08101-4-Q1 S08105-1-Q1 S03859-1-A SP21669249 R Border TOL BB B B B B SP21669332 R Border TOL B B B — B B SP21669615 R Border TOL BB B B B B SP21669616 R Border TOL B B B B B B SP21669670 R Border TOL BB B B B B SP21669503 L Border SEG H H H H H H SP21669458 R Border SUS AA A A A A SP21669760 R Border SUS A A A A A A

TABLE 14D Marker Sample Comment Phenotype S08010-1-Q1 S08010-2-Q2SP21669249 R Border TOL H H SP21669332 R Border TOL H H SP21669615 RBorder TOL B B SP21669616 R Border TOL H H SP21669670 R Border TOL B BSP21669503 L Border SEG H H SP21669458 R Border SUS H H SP21669760 RBorder SUS H H

Example 7 SNP Haplotype Association Analysis

Association analysis of SNP haplotypes across the PPO QTL regionprovides an independent method of validating the PPO interval. From thepanel of susceptible and tolerant lines used to identify SNPs forTaqman® probe development, 235 SNPs from 49 amplicons were identified inthe vicinity of the closely linked marker S03859-1-A. The resulting SNPhaplotype data was analyzed to identify an interval in which all of thehaplotypes from the susceptible and tolerant lines co-segregated witheach other (Table 15).

TABLE 15 SNP haplotype association analysis of the PPO herbicidetolerance QTL interval. Perfect association between haplotype andphenotype between amplicons Gm19_1563k1 and Gm19_1618k2 defines the QTLinterval. GEID Amplicon 1563k1 1563k1 1563k1 1563k1 1618k2 1618k2 627002TOL (PPO) G G A C * C 3911338 TOL (PPO) G G A C * C 1564727 TOL (PPO) GG A C * C 4230314 TOL (PPO) G G A C * C 4135359 TOL (PPO) G G A C * C4611588 TOL (PPO) G G A C * C 1590166 TOL (PPO) G G A C * C 3395451 TOL(PPO) G G A C * C 2322432 TOL (PPO) G G A C * C 4520632 TOL (PPO) G G AC * C 632343 TOL (PPO) G G A C * C 1770139 TOL (PPO) G G A C * C 3587853TOL (PPO) G G A C * C 4553991 TOL (PPO) G G A C * C 5183219 TOL (PPO) GG A C * C 2636517 TOL (PPO) G G A C * C 3495695 TOL (PPO) G G A C * C1737165 SUS (PPO) A * G T A A 1653063 SUS (PPO) A A 4501774 SUS (PPO)A * G T A A 7589905 SUS (PPO) A * G T N A 4832982 SUS (PPO) A * G T N A2839548 SUS (PPO) A * G T 3958440 SUS (PPO) A * G T A A 6116656 SUS(PPO) A * G T A A

Although it is difficult to definitively define the co-segregatingregion, it can conservatively be estimated to reside between ampliconsPPO_Gm19_(—)1563k1 and PPO_Gm19_(—)1618k2. Within the borders defined bythese loci, there are 38 SNP differences that are shared between all ofthe susceptible lines compared to all the tolerant lines. This intervalis approximately the same ˜56 kb interval identified by fine-mapping.

It will be apparent to those of skill in the art that it is not intendedthat the invention be limited by such illustrative embodiments ormechanisms, and that modifications can be made without departing fromthe scope or spirit of the invention, as defined by the appended claims.It is intended that all such obvious modifications and variations beincluded within the scope of the present invention as defined in theappended claims. The claims are meant to cover the claimed componentsand steps in any sequence which is effective to meet the objectivesthere intended, unless the context specifically indicates to thecontrary.

All publications referred to herein are incorporated by reference hereinfor the purpose cited to the same extent as if each was specifically andindividually indicated to be incorporated by reference herein.

1-11. (canceled)
 12. A kit for selecting at least one soybean plant bymarker assisted selection of a quantitative trait locus associated withtolerance to herbicides that inhibit protoporphyrinogen oxidase functioncomprising: a. chemically synthesized or chemically modified primers orprobes for detecting at least one tolerance-associated marker locus,wherein the tolerance-associated locus is selected from the groupconsisting of S08102-1, S08103-1, S08104-1, S08106-1, S08107-1,S08109-1, S08110-1, S08111-1, S08115-2, S08117-1, S08119-1, S08116-1,S08112-1, S08108-1, S08101-4, S08101-1, S08101-2, S08101-3, S08105-1,S08118-1, S08010-1, S08010-2, S60167, P10649C, S00224-1, P5467, S04867-1and S03859-1; and b. instructions in using the primers or probes fordetecting the marker loci and correlating the loci with predictedprotoporphyrinogen oxidase inhibitor tolerance. 13-32. (canceled) 33.The kit of claim 12, wherein the tolerance-associated locus is detectedusing chemically synthesized or chemically modified primers or probesfor a marker selected from the group consisting of S08102-1, S08103-1,S08104-1, S08106-1, S08107-1, S08109-1, S08110-1, S08111-1, S08115-2,S08117-1, S08119-1, S08116-1, S08112-1, S08108-1, S08101-4, S08101-1,S08101-2, S08101-3, S08105-1, S08118-1, S08010-1, S08010-2, S60167-TB,P10649C-3, S00224-1, P5467-1 and S04867-1 and S03859-1.
 34. The kit ofclaim 12, wherein the primers comprise a primer pair selected from thegroup consisting of: a. SEQ ID NO: 34 and SEQ ID NO: 35; b. SEQ ID NO:38 and SEQ ID NO: 39; c. SEQ ID NO: 42 and SEQ ID NO: 43; d. SEQ ID NO:50 and SEQ ID NO: 51; e. SEQ ID NO: 54 and SEQ ID NO: 55; f. SEQ ID NO:62 and SEQ ID NO: 63; g. SEQ ID NO: 66 and SEQ ID NO: 67; h. SEQ ID NO:70 and SEQ ID NO: 71; i. SEQ ID NO: 78 and SEQ ID NO: 79; j. SEQ ID NO:86 and SEQ ID NO: 87; k. SEQ ID NO: 94 and SEQ ID NO: 95; l. SEQ ID NO:82 and SEQ ID NO: 83; m. SEQ ID NO: 74 and SEQ ID NO: 75; n. SEQ ID NO:58 and SEQ ID NO: 59; o. SEQ ID NO: 30 and SEQ ID NO: 31; p. SEQ ID NO:18 and SEQ ID NO: 19; q. SEQ ID NO: 22 and SEQ ID NO: 23; r. SEQ ID NO:26 and SEQ ID NO: 27; s. SEQ ID NO: 46 and SEQ ID NO: 47; t. SEQ ID NO:90 and SEQ ID NO: 91; u. SEQ ID NO: 98 and SEQ ID NO: 99; v. SEQ ID NO:110 and SEQ ID NO: 111; w. SED ID NO: 1 and SEQ ID NO: 2; x. SEQ ID NO:5 and SEQ ID NO: 6; y. SEQ ID NO: 10 and SEQ ID NO: 11; and z. SEQ IDNO: 14 and
 15. 35. The kit of claim 12, wherein the probes are selectedfrom the group consisting of SEQ ID NOs: 7, 8, 9, 12, 13, 16, 17, 20,21, 24, 25, 28, 29, 32, 33, 36, 37, 40, 41, 44, 45, 48, 49, 52, 53, 56,57, 60, 61, 64, 65, 68, 69, 72, 73, 76, 77, 80, 81, 84, 85, 88, 89, 92,93, 96, 97, 108, 109, 112, and 113.